Genetic traits for improved photosynthesis and food production and pathogen resistance in plants

Abstract

By engineering genetic traits associated with photosynthesis in plants, transgenic plants with increased crop yield can be generated, which will ultimately lead to increased food production in the field. To obtain beneficial genetic traits for enhanced photosynthesis, accelerated evolution is applied under salt stress in a cyanobacteria species, Synechococcus elongatus PCC 7942, by conditionally suppressing its mismatch repair system. Through high-throughput screening for biomass accumulation and sucrose production, it is discovered that specific amino acid changes in the pre-Dl protein of Photosystem II (PSII), encoded by the psbA genes, can improve efficiency of photosynthesis under salt stress, as well as pathogen resistance. Next, the same amino acid changes were made in Arabadopsis and proved that photosynthetic efficiency (measured via growth rate) is also increased in this model vascular plant.

Description

Attorney Docket no.10457-590PC0 GENETIC TRAITS FOR IMPROVED PHOTOSYNTHESIS AND FOOD PRODUCTION AND PATHOGEN RESISTANCE IN PLANTS FEDERAL FUNDING NOTICE

[0001] The invention was made with United States Government support under Grant No.2414925 awarded by National Science Foundation. The U.S. government has certain rights in the invention. BACKGROUND 1. Field

[0002] The present invention relates to improving crop yield, more particularly to generating transgenic crop plants with increased photosynthesis efficiency, by engineering genetic traits associated with robust photosynthesis in plants, which will ultimately lead to increased food production. 2. Background

[0003] Human population growth and shrinking farmlands place huge pressure on crop productivity to meet the increasing global food demands. It is projected that crop productivity needs to increase ca. 50-70% by 2050 to meet the global food demand for a population of nearly 9.7 billion. There are several factors determining the crop yield based on the crop yield equation, i.e., photosynthetic solar energy (S), its interception efficiency (^^), conversion efficiency (^c), and partition efficiency (^p)2. Since the last green revolution, all factors except for the photosynthesis conversion efficiency have been nearly maximized to improve crop productivity3. Improving photosynthetic conversion efficiency has thus been proposed as a potential solution to further increase crop productivity in the next few decades.

[0004] Photosynthesis converts solar energy into chemical energy stored in biomass and is fundamental to life on Earth. The theoretical maximum of photosynthetic conversion efficiency from solar energy to biomass ranges from about 4-6% in plants to 9% in microalgae1. However, the average observed efficiencies are typically less than 1% in plants and up to 3% in algae cultivated in photobioreactors2,3, motivating substantial efforts to enhance photosynthetic conversion efficiency to increase crop productivity and develop carbon capture technologies4-6.Attorney Docket no.10457-590PC0 As the last parameter to potentially improve crop yield2, this significant gap between the observed efficiency and the theoretical maximum offers great opportunities to further improve crop productivity. Despite its importance, enhancing photosynthetic efficiency remains a challenging task, often complicated by evolutionary trade-offs developed over billions of years.

[0005] There has been constant debate among crop scientists and photosynthesis researchers about the effectiveness of increasing crop yield through photosynthesis improvement. Plant productivity data either in the greenhouse or in the field shows mixed results for yield improvement through photosynthesis enhancement.

[0006] Key to enhancing carbon fixation and overall photosynthesis in natural settings is the improvement of photosynthesis recycling kinetics. Oxygenic phototrophs, which use tandem photosystems to drive the Calvin-Benson-Bassham (CBB) cycle, are nearly at their theoretical limit for photosynthetic energy conversion efficiency1. Environmental fluctuations frequently disrupt the balance between energy production and consumption7, leading to suboptimal photosynthetic energy conversion. Strategies such as accelerating the recovery of non- photochemical quenching (NPQ) have been shown to improve carbon fixation over a growing season thus enhancing crop yield8. Under abiotic stresses, plants also actively repress growth to maximize survival through stress-triggered cell signaling9. This stress-growth trade-off reduces photosynthetic efficiency10,11, leading to lower crop productivity. However, increasing evidence suggests that improved growth under stress is possible12-14, and such phenotypes can be explained by Pareto optimality15, where certain genetic traits confer the best tradeoff among multiple conflicting tasks.

[0007] Cyanobacteria possess unique genetic traits, such as the gene encoding a bifunctional fructose-1,6-bisphosphatase / sedoheptulose-1,7-bisphosphatase, which has been successfully engineered into plants to enhance photosynthesis and growth16,17. Recent directed evolution studies further identified new genetic traits for improved cyanobacterial growth under high light or combined high light and temperature stress18,19, expanding the potential to optimize photosynthesis by leveraging these beneficial traits.

[0008] Traditional directed evolution methods, however, typically rely on a binary growth / no growth phenotype for isolating mutants, which limits their application to extreme or lethal stress factors. Additionally, long-term evolution experiments tend to optimize traits for stress response, often at the expense of reduced growth under normal conditions18. Salt stress inhibits photosystemAttorney Docket no.10457-590PC0 II (PSII) repairs in cyanobacteria and is a common stress factor for plant growth20,21. Moderate salinity can lead to severe yield losses across a range of crop species22. Identifying beneficial genetic traits in response to sublethal salt stress necessitates methodologies capable of capturing the intricate dynamics of cellular responses to such stressors. SUMMARY

[0009] It is the goal of this disclosure to improve plant photosynthetic conversion efficiency by leveraging beneficial genetic traits or knowledge from cyanobacteria for increased biomass yield and productivity.

[0010] In this disclosure, in vivo accelerated evolution coupled with a high-throughput screening system was employed to identify genetic traits that enhance photosynthesis under salt stress in cyanobacteria. It is hypothesized that a vast array of beneficial genetic traits for photosynthesis improvement is often obscured by simple mutations such as single nucleotide polymorphisms (SNPs) in cyanobacterial genomes, which can be revealed through short-term directed evolution.

[0011] In this proof-of-concept study, novel SNPs in the PSII reaction center D1 gene of Synechococcus elongatus PCC 7942 (hereafter S. elongatus) were identified, which are Pareto- front traits that simultaneously optimize photosynthesis and enhance resistance to both salt and combined salt and light stress. Phylogenetic analysis reveals that one of these mutations is commonly found across cyanobacteria and red algae, while others are rare or even absent in the database, suggesting broad significance of these identified mutations in adapting photosynthesis to environmental stresses. This methodology provides a powerful tool for uncovering novel genetic traits that enhance photosynthesis under stress or other environmental perturbations, with the potential to improve crop yields through translational research.

[0012] In one aspect, genetic traits associated with photosynthesis improvement in cyanobacteria under fluctuating environmental conditions were identified. In this disclosure, salt was applied as the stressor for the directed evolution experiment, and gene targets for photosynthesis improvement were identified. One of the most interesting candidates is the mutated D1 protein, the essential reaction center protein in photosystem II (PSII). A previous study showed that salt stress can inhibit transcription and translation of D1 protein during photodamage repair inAttorney Docket no.10457-590PC0 Synechocystis sp. PCC 680321. The D1 mutations found in this disclosure thus suggest strong associations between stressors and genetic mutations in the directed evolution experiment. Applying other stress conditions such as fluctuating light and heat stress in the mutagenesis experiment could help identify more genetic mutations associated with biomass increase.

[0013] In another aspect, how C terminus extension of D1 protein contributes to photosynthesis improvement in cyanobacteria was determined. In this disclosure, shared D1 mutations were found in several candidate strains. These mutations are all localized to the C terminus extension region of the D1 protein. D1 protein is a core PSII subunit and is subjected to photodamage during photosynthesis charge separation. Frequent D1 replenishment to PSII is required to avoid photoinhibition. However, D1 maturation in PSII requires the cleavage of 16 amino acids from the carboxyl terminus by a carboxyl-terminal processing protease (CtpA)31.

[0014] In another aspect, missense mutation pD1 proteins and newly evolved genetic traits are introduced as alternatives from cyanobacteria to plants for improved photosynthetic efficiency and biomass yield.

[0015] In summary, a key aspect of enhancing photosynthesis is improving its recycling kinetics, enabling swift resumption of photochemical quenching following environmental disruptions or stress. Salt stress exacerbates high light stress in cyanobacteria and leads to severe yield losses in crop plants. Genetic traits that confer salt tolerance without compromising photosynthetic performance are essential for improving photosynthesis under these conditions. To obtain beneficial genetic traits for enhanced photosynthesis under salt stress, accelerated evolution is applied in Synechococcus elongatus PCC 7942 by conditionally suppressing its methyl-directed mismatch repair system. Screening over 10,000 mutants, eight strains with increased biomass or sucrose productivity under salt stress were isolated. Genome sequencing revealed an average of 8- 20 single nucleotide polymorphisms (SNPs) or indels per genome.

[0016] Notably, mutations in the photosystem II (PSII) reaction center D1 gene, resulting in the amino acid changes L353F, I358N, and H359N at the carboxyl terminus of the pre-D1 (pD1) protein, improve photosynthesis under salt and combined salt and light stress by potentially accelerating D1 maturation during PSII repair. Phylogenetic analysis of pD1 across cyanobacteria and red algae highlights the broad significance of these adaptive genetic traits, underscoring the importance of leveraging evolutionary insights to improve photosynthesis under stress or fluctuating environments. For the application of the mutation of pD1 protein to field crop, it isAttorney Docket no.10457-590PC0 demonstrated here that the introduction of such mutation into Arabidopsis utilizing Agrobacterium / T-DNA improved plant mass as well as root hair growth.

[0017] The results here support the mission of sustaining conventional, organic, urban food, and agricultural and natural systems”, which includes “genetic improvement of plant”. The outcome of the disclosure not only improves the understanding of robust photosynthesis mechanisms but also provides a variety of beneficial genetic traits to increase crop productivity. This will serve as a novel and effective plant breeding strategy that bears long-term positive impact on the sustainability of U.S. as well as global agriculture and food systems.

[0018] In one embodiment, a transgenic plant with improved photosynthesis efficiency is presented, which overexpresses at least one variant of pre-D1 (pD1) protein. The plant comprises cells comprising at least one variant of psbA1 gene encoding the variant pD1 protein, and the variant psbA1 gene is a mutated psbA1 gene having at least one point mutation in a wild type psbA1 gene from cyanobacterium, Synechococcus elongates, and optionally from S. elongates PCC 7942 strain. The wild type psbA1 gene from S. elongatus PCC 7942 comprises a nucleotide sequence of SEQ ID NO:1 encoding a wild type pD1 protein, which comprises an amino acid sequence of SEQ ID NO:2. A variant psbA1 gene having at least one point mutation encodes a variant pD1 protein having at least one missense mutation for amino acid substitution in the carboxyl terminus region near the site to be cleaved by the protease CtpA or near the end of the pD1. In a certain embodiment, the variant psbA1 gene encodes a variant pD1 protein having one or more of the following point mutations,I358N, T354P, H359N, L353F, or T354A.

[0019] In a certain embodiment, the variant psbA1 gene having a point mutation comprises a nucleotide sequence of SEQ ID NO:3, which encodes a variant pD1 protein having a missense mutation of I358N, and wherein the pD1 I358N comprises an amino acid sequence of SEQ ID NO:4.

[0020] In a certain embodiment, the variant psbA1 gene having a point mutation comprises a nucleotide sequence of SEQ ID NO:5, which encodes a variant pD1 protein having a missense mutation of T354P, and wherein the pD1 T354P comprises an amino acid sequence of SEQ ID NO:6.

[0021] In a certain embodiment, the variant psbA1 gene having a point mutation comprises a nucleotide sequence of SEQ ID NO:7, which encodes a variant pD1 protein having a missenseAttorney Docket no.10457-590PC0 mutation of H359N, and wherein the pD1 H359N comprises an amino acid sequence of SEQ ID NO:8.

[0022] In a certain embodiment, the variant psbA1 gene having a point mutation comprises a nucleotide sequence of SEQ ID NO:9, which encodes a variant pD1 protein having a missense mutation of L353F, and wherein the pD1 L353F comprises an amino acid sequence of SEQ ID NO:10.

[0023] In a certain embodiment, the variant psbA1 gene having a point mutation comprises a nucleotide sequence of SEQ ID NO:11, which encodes a variant pD1 protein having a missense mutation of T354A, and wherein the pD1 T354A comprises an amino acid sequence of SEQ ID NO:12.

[0024] In a certain embodiment, the variant psbA1 gene has 2, 3, or 4 point mutations, which encodes a variant pD1 protein having 2, 3, or 4 missense mutations, and wherein the missense mutations are at least two selected from L353F, I358N, H359N, T354P (or T354A), and optionally L353F+T354A+I358N.

[0025] In some embodiments, the plant cells comprising at least one variant of psbA1 gene further comprises ctpA gene comprising a nucleotide sequence of SEQ ID NO:13 encoding CtpA7942 comprising an amino acid sequence of SEQ ID NO:1, and wherein the ctpA gene is from S. elongates PCC 7942 strain.

[0026] In some embodiments, the transgenic plant is a crop plant or Arabidopsis. In addition, a seed and an explant that produces the aforementioned transgenic plant are within the scope of the invention.

[0027] In certain embodiments, a DNA fragment comprises a variant of psbA1 gene having a point mutation. The variant psbA1 gene comprises a nucleotide sequence selected from SEQ ID NOs:3, 5, 7, 9, and 11, which encodes a variant pD1 protein having a missense mutation selected from I358N, T354P, H359N, L353F, or T354A.

[0028] In certain embodiments, a DNA fragment comprises a variant of psbA1 gene having at least two point mutations, which encodes a variant pD1 protein having at least two missense mutations selected from I358N, T354P, H359N, L353F, or T354A.

[0029] The aforementioned DNA fragment may comprise an inducible- or constitutively active promoter before the 5’ region of the psbA1 gene and a terminator sequence after the 3’ region of the psbA1 gene. The DNA fragment may be a T-DNA fragment.Attorney Docket no.10457-590PC0

[0030] In certain embodiments, a plasmid comprises the aforementioned DNA fragment.

[0031] In some embodiments, Agrobacterium comprises the aforementioned DNA fragment or the plasmid. The Agrobacterium may optionally comprise more than one kind of plasmid, and each kind of plasmid comprises a DNA fragment comprising one selected from SEQ ID NOs:3, 5, 7, 9, and 11. The Agrobacterium is optionally Agrobacterium tumefaciens.

[0032] In some embodiments, cyanobacteria comprise aforementioned DNA fragment or the plasmid for biofuel and alcohol production. The cyanobacteria may optionally comprise more than one kind of plasmid, and each kind of plasmid comprises a DNA fragment comprising one selected from SEQ ID NOs:3, 5, 7, 9, and 11. The cyanobacteria are the bacteria belonging to the order of Synechococcales. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1. Schematic representation of energy conversion in photosynthesis. About 2 / 3 of the photon energy is lost in light reactions whereas carbon fixation conserves ~90% of energy in ATP / NADPH.

[0034] Figure 2. Construction of a hypermutator strain for directed evolution in S. elongatus. a. Schematic representation of mutagenesis controlled by nitrogen sources. Ammonium represses the nirA promoter by inhibiting its activator, NtcA. b. Spot assay. An equal amount of mSe0 cells (OD730nm= 1) were diluted in triplicate onto BG11 agar plates supplemented exclusively with either nitrate or ammonium. Cyanobacteria in ammonium medium exhibit higher mutation rates, as evidenced by a reduced number of viable cells. c. RT-qPCR of mutS expression. RT-qPCR was conducted on cells cultivated for 24 hours in both BG11 (NO3-) and BG11 (NH4+) media. The relative expression of mutS was quantified against the reference gene rnpB. Error bars represent the standard deviation from three biological replicates, each with two technical replicates. Statistical significance was determined using a two-tailed Student’s t-test. ** p < 0.01, *** p < 0.001.

[0035] Figure 3. Overview of the high-throughput screening method based on short-term accelerated evolution to obtain Pareto-front traits for enhanced photosynthesis under salt stress. The hypermutator strain mSe0 is subject to three rounds of mutagenesis in BG11(NH4+) for a period of 4 days each round. Following each round, mutants are isolated on BG11(NO3-) agar plates to maintain mutations. Individual mutants are then subjected to high-throughput screeningAttorney Docket no.10457-590PC0 in 96-well microplates to assess both biomass (measured by optical density at 730 nm, OD730nm) and / or sucrose productivity. The final elite strain from each round serves as the starting point for the subsequent round of mutagenesis. The Pareto-front traits endow cyanobacterial mutants with diverse adaptive capabilities for biomass accumulation and stress response.

[0036] Figure 4. High-throughput screening of mSe3 mutants. The top 1% of mutants with the highest standardized residuals of sucrose productivities were selected as the SPM candidates (green), and top 0.25% of mutants with the highest OD730nmwere selected as the BAM candidates (light green). A green solid line represents the fitted linear model based on the mSe0 control. The green dashed line represents two standard deviations (2σ) from the mean of sucrose productivity. The inset shows FGM candidates (medium green) representing the top 0.25% in population size fold change after a 54-hour incubation (24 h normal growth followed by 30 h growth with salt stress).

[0037] Figure 5. Growth phenotype validation in 96-well cell culture plates. a. Population size fold change was normalized against the mean fold change of wild type S. elongatus for each batch to minimize the batch effects. The biomass validation with 12 replicates (4 replicates × 3 batches) reached a statistical power of 89.7%. b. Sucrose production validation based on 96-well cell culture plates. The validation with 16 replicates (8 replicates × 2 batches) reached a statistical power of 98%.

[0038] Figure 6. Mutation in BAM, FGM, and SPM elite mutants. a. Mutations from eight mSe3 strains and wild-type S. elongatus with allele frequencies above 0.25 are displayed. Mutations are classified into indels and SNPs in either coding sequences (CDS) or non-coding DNA (ncDNA) regions. b. Mutations located on CDS are shown, with shades of blue indicating allele frequency (AF). Grey shows sites with either the reference alleles or mutations with AFs of < 0.25. c. O2 evolution revealed slight difference among different mutants.

[0039] Figure 7. Interaction of pD1 and CtpA simulated by AlphaFold335. CtpA is depicted using a surface representation, while the carboxyl terminus tail of wild-type pD1 (top row) and mutated pD1 (bottom row) are shown in ribbon format with mutated residues displayed in stick structures. Dark cyan represents hydrophilic surfaces, and dark goldenrod denotes lipophilic surfaces. Nearby amino acid residues around the mutation site are annotated.

[0040] Figure 8. Validation of pD1 mutations under salt and moderate light stresses. Schematic representation of pD1 mutations and competitive assembly of D1 during PSII repair.Attorney Docket no.10457-590PC0

[0041] Figure 9. Validation of stress resistance. Transformants with mutations L353F, I358N, H359N, and combined L353F+T354A+I358N showed superior biomass accumulation compared with the psbA1WTcontrol under 150 mM NaCl and combined stress of salt (150 mM) and moderate light (110 μE·m−2·s−1) stress. Note that this light intensity leads to chlorosis of cyanobacteria in 96-well culture plates, suggesting moderate light stress. Transformants with L353F and combined L353F+T354A+I358N mutations outperformed the psbA1WTcontrol under 110 μE·m−2·s−1light stress alone. For statistical analysis; each experiment included 16 replicates per transformant, with statistical significance determined using an unpaired Student’s t-test (*p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

[0042] Figure 10. The pD1 L353F mutation confers improved growth and salt stress. a. Growth dynamics of psbA1WTand psbA1L353Ftransformants were monitored in triplicate in multi- cultivators. psbA1WTis shown in yellow and psbA1L353Fin green, with increasing shading intensities corresponding to increasing salt concentrations (0 mM, 150 mM, 300 mM). Data points represent the mean of three independent biological replicates, with ribbons indicating one standard deviation from the mean. Statistical analysis was performed using an unpaired Student’s t-test based on OD730nm measurements at 30 h. b. Phycobilin content was estimated using full light spectrum scanning. Samples from late-log phase cells under different salt concentrations (0 mM, 150 mM, 300 mM) and the light intensity of 300 μE·m−2·s−1were analyzed in triplicate. Absorbance spectra ranging from 300 nm to 700 nm were recorded with a microplate reader, averaged from biological triplicates, and normalized to biomass (A730). Oxygen evolution rates were measured under growing light (c. 300 μE·m−2·s−1) and saturating light (d. 4,000 μE·m−2·s−1) conditions. Statistical significance was assessed using an unpaired Student’s t-test (*p < 0.05, ** p < 0.01).

[0043] Figure 11. The predicted structure of S. elongatus pD1 protein using Alphafold. The red arrow indicates the Ala344 cleavage site at the C terminus extension.

[0044] Figure 12. Schematic representation of pD1 supplementation in plants for improved photosynthesis and growth

[0045] Figure 13 represents T1 generation Arabidopsis plants transformed with only psbA1 gene from wild-type S. elongatus PCC 7942. Transformants are healthy with some plants showing elongated leaves.Attorney Docket no.10457-590PC0

[0046] Figure 14 represents T1 generation Arabidopsis plants transformed with psbA1 and ctpA genes from wild-type S. elongatus PCC 7942. Some plants show yellowing leaves or elongated leaves.

[0047] Figure 15 represents T1 generation Arabidopsis plants transformed with only mutated psbA1 gene (L353F) from S. elongatus mutant.

[0048] Figure 16 represents T1 generation Arabidopsis plants transformed with mutated psbA1 gene (L353F) along with its protease CtpA from S. elongatus.

[0049] Figure 17. a. Root length of T3 homozygous transgenic plants. Two independent T3 lines are shown for both Arabidopsis plants transformed with the wild-type and mutated psbA1 gene (L353F) from S. elongatus. b. Representative plant root images corresponding to the statistical analysis in Figure 17a.

[0050] Figure 18. Basal resistance to the pathogen Pseudomonas syringae pv. maculicola ES4326 (Psm). To assess basal resistance to Psm, two leaves from each 4-week-old plant were infiltrated with a Psm suspension (OD600 = 0.0003). Bacterial populations were measured 3 dpi to evaluate pathogen presence. The results show that all transgenic lines have higher basal resistance compared to the Col-0 wild type. In particular, the psbA1L353 -4 line showed the highest resistance to the pathogen Psm. Detailed Description 1. Overview

[0051] Increased crop yield is determined by the average effective photosynthetic energy conversion over a growing season. In nature and agricultural practices, plants experience constant environmental changes (e.g. fluctuating light and temperature) and other biological perturbations (e.g. insects), which disrupt the balance between energy production in light reactions and energy consumption by the carbon metabolism. The constant disruption and reinitiation of energy conversion process is compounded over a growing season, leading to lower than the theoretical energy conversion efficiency to biomass. It is thus challenging to account for all the variables in environmental perturbations that could affect the photosynthesis engineering efforts in plants. This helps explain the inconsistent results observed in the field for crop yield improvement through photosynthesis engineering.Attorney Docket no.10457-590PC0

[0052] During evolution, plants have also evolved complex photosynthesis chemistry and regulatory mechanisms to help reinitiate photosynthesis following environmental perturbations. Here, robust photosynthesis refers to these collective chemistry efforts in increasing overall photosynthetic conversion efficiency. Engineering photosynthesis chemistry thus can lead to further kinetic improvement of photosynthesis reinitiation, resulting in higher energy conversion efficiency and plant biomass yield.

[0053] Photosynthesis limitation lies in its kinetic operation rather than the max efficiency. The limitation of photosynthesis can be understood from the fundamental mechanisms of light energy conversion process. In the light reactions, nine photons (one photon used for the cyclic electron flow) are absorbed to form three ATP and two NADPH, losing roughly two thirds of the energy. These chemical energies are just enough to drive one cycle of the Calvin-Benson-Bassham (CBB) cycle reactions, reducing one molecule of CO2 into 1 / 6 of the glucose molecule. Interestingly, the CBB cycle conserves almost 90% of the energy in the ATP and NADPH into biomass (Fig.1). By design, nature has evolved a highly efficient photosynthetic energy conversion process in plants, leaving little space for photosynthesis pathway redesign. Improving photosynthesis thus largely relies on improving its kinetic operations to ensure continuous energy flux from photon to biomass.

[0054] A large repertoire of beneficial genetic traits is required for future plant breeding to improve photosynthesis and crop yield.

[0055] To further improve crop productivity, an expansion of beneficial genetic traits is urgently needed for plant breeding. However, it is challenging to conduct this effort in plants directly. Cyanobacteria serve as ideal chassis to screen new genetic mutations associated with increased photosynthetic carbon assimilation. Because of shared photosynthesis chemistry, many of these genetic traits are transferrable from cyanobacteria to plants.

[0056] Increasing evidence in cyanobacteria suggests that cellular metabolism can be altered through simple mutations such as single nucleotide polymorphisms (SNPs) and INDELs. In order to obtain beneficial mutations for photosynthesis, an in vivo hypermutator strain in the cyanobacterium S. elongatus PCC 7942 was constructed based on a previous design. In this mutator strain, the native promoter of the mutS gene encoding a core mismatch repair (MMR) protein was replaced with an inducible promoter regulated by nitrogen sources (Fig. 2). When grown in ammonium-containing medium, S. elongatus turns into a mutator strain due to the repression of the mutS gene expression. External stress can then be applied to direct the evolutionAttorney Docket no.10457-590PC0 trajectory. Following the directed evolution, desirable mutations can be maintained by simply switching into the nitrate-containing medium. Short-term (days) mutation experiments were applied to obtain manageable numbers of mutations for downstream validation experiments. One limitation in long-term (months) evolution experiments is the mutations were identified under steady state growth, thus likely resulting in mutations for stress resistance (e.g., high light / temperature / salinity) rather than photosynthesis improvement. Also, long-term evolution experiments could further steer evolution trajectory toward stress response rather than photosynthetic growth. This platform can be utilized to identify manageable genetic mutations for the follow-up reverse engineering efforts.

[0057] In the meantime, it requires a high-throughput screening platform to capture genetic mutations early in the evolution among a large cyanobacterial population. Salt stress can affect photosystem repairs in cyanobacteria and is a common stress factor for plant growth. In the experiments, the mutator cells were subjected to three rounds of mutagenesis by applying increasing salt concentrations as the external stress factor. Individual cyanobacterial mutants were inoculated into 96-well plates and incubated in an in-house growth chamber with salt added in the later stage of the growth. Both biomass and sucrose productivity were used as screening targets for improved photosynthetic efficiency. Meanwhile, the wild type S. elongates was introduced in the same manner to serve as the control. A regression-based model was used to identify candidate strains that were superior sucrose producers, while terminal biomass or biomass fold change were monitored by measuring the optical density. In summary, top candidates in three classes of mutants: Sucrose-Producing Mutants (SPMs), Biomass-Accumulating Mutants (BAMs), and Fast- Growing Mutants (FGMs) were isolated for subsequent validation assays (Fig. 4).

[0058] Following preliminary screening, the above top candidate strains were selected for validation assays to confirm whether they are genuine mutants with statistically different traits in either biomass or sucrose production compared with the wild type. Power analysis was applied to calculate the ideal numbers of replicates to be included in the validation assay. Ultimately, six mutants associated with biomass accumulation and two mutants for sucrose production were identified as elite mutants (Fig. 5) and were selected for genome sequencing. The bidirectional distribution of these mutants supports the hypothesis that cyanobacterial cells have diverse strategies to combat environmental stress such as salt, i.e. other than sucrose which is a compatible solute for salt stress. Selecting terminal phenotype in a long-term evolution experiment thus mightAttorney Docket no.10457-590PC0 not be beneficial for identifying genetic traits associated with photosynthesis improvement. The large variations in the validation assay further indicate that mutations are likely not segregated fully due to the polyploidy nature of the genome in S. elongatus (Fig. 5).

[0059] High coverage (>1000x) genome sequencing of elite strains revealed several interesting genetic mutations potentially contributing to robust photosynthesis. Overall, there are on average ~10 SNPs or INDELs found in these elite mutants. These mutations could be further narrowed down by genetic analysis to provide manageable gene targets for reverse engineering in the wild type.

[0060] Among these mutations, the most interesting mutations were found in the psbA1 gene, encoding the core photosystem II reaction center D1 protein29. D1 protein is subjected to constant photodamage and must be replaced every few minutes to ensure optimal photosynthesis in cyanobacteria and other oxygenic phototrophs30. The D1 gene in S. elongatus encodes 360 amino acids, termed pre-D1 (pD1). During PSII assembly, the 16 amino acids at the C terminus are cleaved to form the mature D1 (mD1) by the protease CtpA31(Fig. 11).

[0061] In this disclosure, all point mutations in the psbA1 gene were found at the carboxyl terminus extension of the D1 protein (e.g., I358N, T354P, H359N, L353F, or T354A). It is likely that these mutations have led to faster cleavage kinetics during D1 maturation, thus resulting in more time used for effective photosynthesis energy conversion and higher terminal biomass. Understanding the detailed mechanisms thus could facilitate the translational research to engineer alternative D1 in plants for improved photosynthesis and higher carbon yield.

[0062] Summary of Results

[0063] Cyanobacteria emerged over three billion years ago with the development of tandem photosystems to harvest solar energy, leading to the Great Oxidation Event on Earth36. With its current design, photosynthesis operates near its theoretical limit in converting solar energy into chemical energy stored in biomass1. An effective strategy to improve photosynthesis for emerging societal challenges, such as increasing crop yield, is to enhance its recycling kinetics to boost effective photochemical quenching under fluctuating environments37. Over billions of years, evolution has likely accumulated a vast repertoire of beneficial genetic traits among oxygenic phototrophs to adapt photosynthesis to constantly changing environments38,39, with potential Pareto-front genetic traits distributed throughout their genomes.Attorney Docket no.10457-590PC0

[0064] Glimpses of these beneficial genetic traits can be seen embedded in the genomes of cyanobacteria via a few SNP mutations, as evidenced by endowing S. elongatus with a high-light- tolerant phenotype through complementing SNPs found in its close relative, S. elongatus UTEX 297340. Recent directed evolution studies further validate this hypothesis, showing that new SNP traits can be revealed in model cyanobacteria for enhancing photosynthesis under high light or temperature stresses18,19. However, a limitation of applying directed evolution to enhance photosynthesis is its dependence on applying lethal stress factors, thus a binary growth / no growth phenotype can facilitate the isolation of potential beneficial mutants.

[0065] To mitigate this challenge, short-term in vivo accelerated evolution by conditionally suppressing the mutS gene of the MMR system was applied, thereby easily inducing and maintaining SNP mutations for improved photosynthesis under salt stress (Fig.2). Combined with a relatively high-throughput screening system, eight S. elongatus mutants with enhanced biomass or sucrose productivity under salt stress were isolated by screening just over 10,000 mutants (Fig. 3), demonstrating the effectiveness of this approach in generating beneficial SNP mutations for improved photosynthesis.

[0066] The final elite strains contain an average of 8-20 SNPs and indels in their genomes, with the majority being missense mutations in CDSs and substitutions in non-coding regions (Fig. 6). The mutations found in the psbA1 gene, encoding the PSII reaction center D1 protein, were identified as the contributing factor for improved photosynthesis in the mutants. Intriguingly, these mutations were located at the carboxyl terminus of the pD1 protein at positions 353, 358, and 359. The 353 position is adjacent to the early cleavage site Ala352 during pD1 processing32,33, suggesting its potential involvement in altering pD1 cleavage efficiencies. In Synechocystis sp. PCC 6803, replacing Asn359 with either His or Asp resulted in higher vulnerability to photoinhibition34. In S. elongatus, there are three different copies of psbA genes in the genome. The psbA1 gene encodes the D1 with His359 whereas psbA2 / 3 genes encode the D1 with Asn35941, with psbA2 / 3 gene expression induced under high light conditions. These previous findings suggest that Asn359 might be involved in photoprotection under high light or fluctuating light conditions. Interestingly, the mutation at position 359 in this study showed the conversion of His359 to Asn359 in the psbA1 encoded D1, likely improving the D1 maturation process for robust photosynthesis under stress.Attorney Docket no.10457-590PC0

[0067] Mutations of L353F, I358N, and H359N in the pD1 protein confer stress tolerance to salt and combined salt and moderate light stress in S. elongatus (Fig. 8). Further validation using L353F as an example demonstrates both improved photosynthetic kinetics and maximum capacity due to the introduced mutation (Fig. 9). Previous studies in cyanobacteria and algae showed that mutants without the carboxyl terminus extension in D1 have normal PSII activity and growth but compromised competitiveness when co-cultured with the wild type42-44. A later study observed an increase in unassembled D1 protein and a slight increase in sensitivity to photoinhibition in the mutant strain lacking the C terminus extension, suggesting that this region could mediate D1 assembly into PSII34. Additionally, altering the C terminus extension could impact its binding efficiency with CtpA, thus affecting D1 maturation31. These previous studies indicate a strong association between pD1 carboxyl terminus diversity and robust photosynthesis. 2. Definitions

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled person in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0069] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, microbiology, genetics, protein, and nucleic acid methods and techniques described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed through the present specification unless otherwise indicated.

[0070] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / −10% or less, + / −5% or less, + / −1% or less, and + / −0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0071] Reference throughout this specification to “one embodiment”, “some embodiment,” “certain embodiment,” means that a particular feature, structure or characteristic described inAttorney Docket no.10457-590PC0 connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in some embodiment,” or “certain embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may include other various similar embodiments. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0072] The term “nucleotide” as used herein refers to a subunit of a nucleic acid (whether DNA or RNA or an analogue thereof) which may include, but is not limited to, a phosphate group, a 5- carbon sugar group and a nitrogen-containing base, as well as analogs of such sub-units. Other groups (e.g., protecting groups) can be attached to the sugar group and nitrogen-containing base group. It will be appreciated that, as used herein, the terms “nucleotide” will include those moieties which contain not only the naturally occurring purine and pyrimidine bases, e.g., adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U), but also modified purine and pyrimidine bases and other heterocyclic bases which have been modified (these moieties are sometimes referred to herein, collectively, as “purine and pyrimidine bases and analogs thereof”).

[0073] The terms “nucleotide sequence,” or “nucleic acid sequence” as used herein refers to the specific order or arrangement of nucleotides (adenine, guanine, cytosine, and thymine-base in DNA, or uracil base in RNA, represented using the letters A, C, G, and T (or U in RNA)) within a nucleic acid, DNA or RNA, which carries genetic information. The order of some nucleotides dictates the sequence of amino acids in proteins, which in turn determines the structure and function of those proteins. The term “nucleic acid” refers to unmodified RNA or DNA or modified RNA or DNA as well as single and double-stranded DNA, DNA that comprises single and double-stranded regions, single and double-stranded RNA, and RNA that comprises single and double-stranded regions, hybrid molecules between DNA and RNA, which may comprise single stranded DNA or RNA.

[0074] The term “vector” as used herein refers to a nucleic acid molecule capable of transporting a foreign nucleic acid fragment, and the vector nucleic acid is ligated to the foreign nucleic acidAttorney Docket no.10457-590PC0 fragment. It is usually a DNA molecule that is used as a vehicle to carry a particular foreign nucleic acid sequence, usually DNA, into a host / recipient cell where it can be replicated and / or expressed. The vector typically includes features to facilitate the manipulation of DNA as well as a genetic marker for their selective recognition. The most common vectors are DNA plasmids, viruses and artificial chromosomes. Sometimes, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. Some vectors can be autonomously replicated in a host cell (episomal vectors), or others may be integrated into the genome of a host cell and replicated along with the host genome (non-episomal vectors). Integrating vectors typically contain at least one sequence homologous to the bacterial chromosome that allows for recombination to occur between homologous DNA in the vector and the bacterial chromosome. Integrating vectors may also comprise bacteriophage or transposon sequences.

[0075] The term “plasmid” as used herein refers to a double-stranded, covalently closed, circular DNA into which additional DNA segments can be ligated and which can be isolated from bacterial cells. Plasmid exists in its bacterial hosts as extrachromosomal pieces of DNA that vary in size from 1 kb to >200 kb. Most of the plasmids used in molecular cloning have a multiple cloning site (MCS), also called a polylinker, which is a short segment of DNA that contains various restriction enzyme sites, and this is a standard feature of engineered plasmids for the insertion of a foreign DNA. In addition, the plasmid should have an origin of replication (ori) site – usually bacterial origin where DNA replication is initiated, marker genes – antibiotic resistance gene for selection and / or screening with antibiotics, and promoters – usually viral origin for gene expression. It should be small in size so that it can be easily delivered into the host cell. Plasmids do not generally replicate in the host eukaryotic cells. By performing a process of DNA transfection or transformation, a plasmid which contains a gene of interest is efficiently delivered to the cells. Numerous plasmid vectors are commercially available, and the modification thereof for specific cloning strategies is well known to the skilled person in the field.

[0076] Plant diseases that are preventable or treatable in accordance with the present disclosure can result from any pathogen. As used herein, the term “plant pathogen” is to be understood to refer to any organism that causes disease in plants. Illustrative examples include pathogenic bacteria, fungi, molds, parasites such as nematodes and viruses specific to plant species which are responsible for induction of disease symptoms. In a particular embodiment of the disclosure, theAttorney Docket no.10457-590PC0 pathogen is a soil or stubble-borne pathogen. In exemplary embodiments, the pathogen is a bacterial or fungal pathogen.

[0077] As used herein, a "promoter" is defined as a regulatory DNA sequence that is generally located upstream of a gene and capable of binding RNA polymerase to initiate transcription. Some plasmids may comprise more than one RNA polymerase II (pol II) promoters and / or RNA polymerase III (pol III) promoters. A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active / "ON" state), or it may be an inducible promoter (i.e., a promoter whose state, active / "ON" or inactive / "OFF"), which is controlled by an external stimulus, (e.g., the presence of a particular compound or protein).

[0078] As used herein, the term “cyanobacteria” refers to a group of autotrophic (converting abiotic sources of energy into energy stored in organic compounds) gram-negative bacteria, including orders of Chroococcales, Chroococcidiopsidales, Gloeobacterales, Nostocales, Oscillatoriales, Pleurocapsales, Spirulinales, and Synechococcales. Cyanobacteria are characterized by their ability to perform oxygenic photosynthesis using light-harvesting pigments such chlorophyll, carotenoids, and phycobilins to convert light energy to chemical energy. Cyanobacteria have a unique structure of internal membranes, i.e., flattened sacs called thylakoids where photosynthesis occurs. In photoautotrophic eukaryotes such as algae and plants, photosynthesis is performed in chloroplasts, that contain chlorophyll, giving plants their green color. Chloroplasts are considered to be intracellular endosymbiotic photosynthetic cyanobacteria.

[0079] As used herein, the term "plant" refers to the entire plant including all its parts (e.g., roots, stems, leaves, and flowers), plant parts, plant cells, seeds, and progeny thereof. The plants which can be used in the methods of the invention include both monocotyledonous and dicotyledonous plants, including any plant capable of photosynthesis.

[0080] As used herein, the term “crop plant” refers to a plant often grown in large quantities for human or animal use. They can be classified into several groups for specific use purposes. Food crops are plants cultivated for human consumption, like grains, vegetables, fruits, and nuts. Feed crops are plants cultivated for livestock consumption, like hay, oats, and corn. Fiber crops are plants cultivated for textile fibers, ropes, and paper, like cotton, flax, hamper, and bamboo. Oil crops are plants cultivated for their oils, used for cooking, biofuel, and other applications, like soybeans, cottonseed, sunflower seed, canola, rapeseed, and cacao. Ornamental crops are plants cultivated for their aesthetic value for decoration in gardens, homes, and landscapes, like flowersAttorney Docket no.10457-590PC0 and shrubs. Industrial crops are plants cultivated for raw materials in various industries, like rubber and tobacco.

[0081] The term “transgenic plant” as used herein refers to the plant that have been genetically modified by the insertion of a foreign genetic material (gene(s), DNA sequence(s), etc.) into the chromosome of the cell, while a “transformed plant” is the one whose genome has been genetically modified but not necessarily through insertion of a foreign genetic material. A transgenic plant may have an altered chromosomal genome containing a DNA sequence or gene from a different species, which expresses a protein that is not native to the plant. The protein encoded by the gene will confer a particular trait or characteristic to that plant, such as increased photosynthesis efficiency, or enhanced growth rate or biomass accumulation.

[0082] The term “transformation” or “transform” refers to genetic transformation that is a process that involves the introduction and expression of foreign genes in a host organism. This expression can result from the extrachromosomal presence of a foreign gene or DNA sequence in nuclei, which may persist if the introduced a foreign gene or DNA sequence is inserted into the chromosomal genome of the host. Bacterial transformation is a process by which some bacteria take up foreign genetic material (naked DNA) from the environment. For example, in some embodiments, the Agrobacterium or cyanobacteria are transformed with plasmids, using electroporation or heat shock methods.

[0083] Plant transformation is a process by which foreign genetic material (DNA and / or RNA) is introduced into plant cells, tissues, or organs using various methods, including Agrobacterium-, Rhizobium-, or virus-mediated transformation, polyethylene glycol (PEG) or liposome-mediated transformation, electroporation, microinjection, and DNA-coated particles (e.g., gold or tungsten) bombardment using compressed gas or a gene gun. Plant transformation comprises steps of delivery of the DNA (and / or RNA) into plant cells, regeneration into entire fertile plants, and selection of the transformed plant. For Agrobacterium-mediated transformation, Agrobacterium tumefaciens is the most commonly used species.

[0084] Agrobacterium-mediated transformation involves T-DNA transfer and integration. Agrobacterium carries a tumor-inducing (Ti) plasmid, which comprises a portion of DNA, known as T-DNA, which can be transferred into the nucleus of a plant cell, where it can integrate into the genome of the plant. This T-DNA can be designed to contain a gene or DNA sequence of interest as well as selection marker gene to be introduced into the plant. The floral dip transformationAttorney Docket no.10457-590PC0 method is an example of Agrobacterium-mediated plant transformation. In this method, flower buds are dipped into Agrobacterium suspension until they are covered by the suspension. After bacterial infection, the flower buds are wrapped with paper for about 48 h to maintain high humidity. Then, the flowers are maintained for a few weeks until the seeds are produced. The seeds are harvested, dried at room temperature, and germinated in the medium containing specific antibiotic for selecting putative transformed plants. Agrobacterium can also be used to infect protoplasts, which are plant cells with their cell walls removed, optionally in the presence of PEG to stabilize the protoplast membrane and promote DNA uptake. Protoplast can also be transformed with foreign DNA, RNA, or proteins using methods like liposome, PEG-mediated transformation, microinjection, or electroporation without using Agrobacterium.

[0085] As used herein, “explant” or “plant explant” refers to a fragment of plant tissue obtained from any part of the plant, which can be used as a starting material to grow a plant in tissue culture. Some examples of explants are root fragments, shoot fragments, leaf sections, petals, apical buds, or seeds. From there, the explant can be used for regeneration or non-regeneration techniques.

[0086] As used herein, the term “mutant”, “mutated”, or “variant” refers to a nucleic acid sequence or an amino acid sequence having substantial similarity to the sequence of the wild type. A variant comprises a substitution (point mutation) of one or more nucleotides or amino acids at one or more internal sites within the wild type of polynucleotide or polypeptide. A point mutation can be introduced via PCR, also known as site-directed mutagenesis. This mutagenesis method involves designing primers having a desired point mutation as a mismatch within the primer sequence of around 24-30 nucleotides, with the mutation ideally placed in the middle, flanked by 10-15 perfectly matched nucleotides on each side. Generally, a forward and reverse primer pair are used, with one or both primers containing the mutation. 3. Examples of Certain Embodiments

[0087] Through accelerated laboratory evolution in cyanobacteria, several cyanobacteria mutant strains with enhanced photosynthesis efficiency were identified, and their whole genomes were sequenced, revealing beneficial SNP (single nucleotide polymorphism) mutations in psbA1 gene for robust photosynthesis.

[0088] As demonstrated in EXAMPLES, cyanobacteria were transformed with the plasmid comprising a variant of psbA1 gene having one of the point mutations or combination thereof so as to improve photosynthesis efficiency. It can be contemplated to transform the bacteria withAttorney Docket no.10457-590PC0 different combinations of those variants, and this can be done by transforming the bacteria with combination of plasmids, each containing a different variant psbA1 gene, or with the plasmid containing a variant psbA1 gene having multiple point mutations. Such transformation strategies for introducing multi-mutation protein(s) can be applied to crop plants as well as edible seaweed (e.g., nori, wakame, kombu, sea grapes, agar-producing seaweed (e.g., Gracilaria and Gelidiaceae), and chlorella) to increase their photosynthesis and biomass.

[0089] In EXAMPLES, it is also demonstrated that the transformation of cyanobacteria with the variant psbA1 gene(s) having a point mutation showed enhanced sucrose production. Sucrose, a disaccharide made of glucose and fructose, is readily fermentable, and it can be used to produce biofuels, primarily ethanol, as a fuel source or as an additive in gasoline to reduce fossil fuel consumption. Besides ethanol, sucrose can also be used to produce other biofuels, such as natural gas, through a process involving anaerobic fermentation and gasification. Such an increase in carbohydrate accumulation due to improved photosynthesis in the psbA1 variant transformed bacteria can also be expected in biofuel plants (e.g., corn and sugarcane) expressing the psbA1 variant.

[0090] In certain embodiments, photosynthesis-advantageous amino acid substitutions (missense mutations) in the pD1 protein due to point mutations in psbA1 gene are found in the carboxyl terminus region near the site to be cleaved by the protease CtpA or near the end of the pD1, e.g., L353F, I358N, H359N, T354P (or T354A). The mutation positions found in the pD1 protein strongly suggest their involvement in D1 processing by the CtpA protease, and thus the overexpression of CtpA with the missense mutant preD1 protein might enhance the conversion of pre-D1 protein to mature D1 protein (mD1), and repair cycle of the PSII D1 protein damaged by light induced phosphorylation (Fig.16). To make D1 protein repair faster in plant cells, ctpA gene encoding the protease can also be co-expressed with a psbA1 gene variant, and the gene encoding CtpA can be from the S. elongates PCC 7942 strain or different bacterial strains or species or from plants.

[0091] For transgenic plant generation with variant psbA1 genes, plant cells can be transformed using Agrobacterium / T-DNA or other methods mentioned above. Without gene editing (or genome editing), the gene of interest can be expressed from an extra copy of the gene integrated into the plant cell chromosome at a certain site or random site. For such extra gene expression, the gene needs to be cloned with a strong promoter for protein expression into the T-DNA.Attorney Docket no.10457-590PC0

[0092] On the other hand, it can be contemplated to introduce a point mutation to a gene of interest. The gene can be edited utilizing CRISPR-Cas9 (or Cas12) system and a base editing enzyme, e.g., a cytosine base editor (CBE) mediating a C to T change, which introduces a G to A change on the opposite strand; and an adenine base editor (ABE) mediating a A to G change, which introduces a T to C change on the opposite strand. For example, the base-editing enzyme can be fused to- or coupled with an inactivated Cas nuclease to be delivered to the target site, where the endonuclease does not cause double-strand breaks in the DNA. The genes encoding a guide RNA and the fusion protein of a Cas endonuclease and a base editing enzyme can be expressed from the T-DNA. However, it is also possible to introduce base editing without using Agrobacterium / T-DNA system. Ribonuclear protein (RNP) complex comprising a guide RNA, an endonuclease, and base editing enzyme can be delivered to the plant cell by liposome, PEG, electroporation, or other transformation methods.

[0093] In case of gene replacement, for example, for introducing multiple mutations into a gene, steps of removal of the host gene and insertion of a foreign gene are required, and this can also be done using CRISPR / Cas gene editing system. For gene replacement, a donor DNA template fragment comprising the desired gene sequence (e.g., variant psbA1 gene having multiple point mutations) needs to be delivered along with CRISPR / Cas, which can be expressed from a plasmid or DNA fragment comprising genes encoding guide RNA and Cas endonuclease as well as a selection marker for transgenic plant selection after transformation, or which can be delivered as a ribonuclear protein (RNP) complex comprising a guide RNA and a Cas endonuclease using a method of liposome, PEG, electroporation, or other transformation methods. EXAMPLES

[0094] Example 1. Materials and Methods

[0095] 1.1. Strains and culture condition

[0096] The wild-type Synechococcus elongatus PCC 7942 was cultured on BG11 agar plates for isolation and maintained in BG11 liquid media at 25°C under a light intensity of 40 μE·m−2·s−1for routine cultivation. The nirA promoter sequence was amplified from S. elongatus wild type and used to construct pXWO3_nirA plasmid using NEBuilder HiFi DNA Assembly Master Mix. The mSe0 mutator strain, created from pXWO3_nirA transformation, along with mutants derived fromAttorney Docket no.10457-590PC0 directed evolution, were kept in BG11 (NO3-) supplemented with 5 μg / mL kanamycin. During directed evolution experiments, the mutator strain was grown in BG11 (NH4+) supplemented with 10 mM NaHCO3, 5 μg / mL kanamycin, and different concentrations of NaCl (150, 200, 250 mM). S. elongatus harboring the sucrose permease gene cscB was propagated on BG11 agar plates containing 5 μg / mL chloramphenicol and 5 μg / mL kanamycin and cultivated in BG11 liquid containing the same concentrations of antibiotics. Escherichia coli DH5α, which carries the NSIII cscB plasmid, was cultured in LB broth with 25 μg / mL chloramphenicol and maintained on LB agar plates supplemented with 50 μg / mL chloramphenicol.

[0097] 1.2. Plasmid transformation into S. elongatus strains

[0098] S. elongatus cultures were grown to an OD730nmof 1.0. One milliliter of each culture was collected and centrifuged at room temperature for 2 minutes at 13,000 rpm. The cell pellet was washed with 1 mL of 10 mM NaCl and centrifuged again. The resulting pellet was then resuspended in 200 µL of BG11 (NO3-) medium. To this suspension, 100 ng of plasmid DNA was added and gently mixed. The mixture was incubated in the dark at 30°C for 4-16 hours with gentle agitation. After incubation, the cultures were plated on BG11 (NO3-) agar plates containing the appropriate antibiotics and incubated at 30°C under light conditions.

[0099] 1.3. Reverse Transcription-Quantitative PCR (RT-qPCR)

[0100] S. elongatus wild type and mSe0 strains at mid-exponential growth phase were harvested by centrifugation. The cell pellets were washed and resuspended in either BG11 (NO3-) or BG11 (NH4+) medium. Biological triplicates of both strains were then inoculated in their respective media and cultured to mid-exponential phase at 30°C under continuous light at 45 μE·m−2·s−1. For RNA isolation, cultures were collected and centrifuged at 4°C, and RNA was extracted using the TRIzol-chloroform-isopropanol method according to the manufacturer's protocol (Fisher Scientific, Pub. No. MAN0001272). RNA was subsequently reverse-transcribed to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad, INST-653 Ver D). For the qPCR analysis, primers for mutS and the reference gene rnpB were designed using Primer 3. Standards and samples were run in technical duplicates in the CFX 96 Connect Real-Time PCR system (BioRad) following the SYBR Green kit protocol. The differential gene expression of mutS was measured by qPCR using CFX Connect software 3.1 employing the ΔΔCq method relative to the ‘reference’ gene rnpB.

[0101] 1.4. Spot assayAttorney Docket no.10457-590PC0

[0102] mSe0 mutants at early-mid log phase were resuspended to an OD730nmof 1 in BG11 (NO3- ) and BG11 (NH4+), respectively. After 1:10 serial dilution, 5 µL aliquots were spotted onto corresponding agar plates supplemented with kanamycin. Plates were incubated at 30°C under a light intensity of 40 μE·m−2·s−1for one week. The survival colonies with the same dilution are results of different mutation rates between nitrate and ammonium conditions.

[0103] 1.5. Directed Evolution

[0104] A single colony from the mSe[i] strain series was grown in BG11 (NO3-) in a 6-well plate at 30°C and 30 μE·m−2·s−1until visibly green. The culture was then scaled up in flasks (1:20 v / v) and grown until the OD730nm reached 0.7-1.0. Cells were harvested by centrifugation at 10,000 rpm for 1 minute at room temperature, washed three times with BG11 (NH4+), and resuspended in BG11 (NH4+) for mutagenesis in a 6-well plate. Cultivation continued for 4 days in a shaking incubator set to 230 rpm, 30 μE·m−2·s−1light intensity, and 30°C, with additions of kanamycin and NaCl. Following mutagenesis, cells were washed three times with BG11 (NO3-), resuspended in 100 µL of BG11 (NO3-), diluted appropriately, and plated on BG11 (NO3-) agar plates supplemented with kanamycin.

[0105] 1.6. Optimization of mSe0 growth in 96-well microplates.

[0106] The mSe0 strain was initially grown in 20 mL of BG11 (NO3-) until its OD reached approximately 0.1. Subsequently, 1 µL of this culture was transferred into each well of a 96-well microplate containing 249 µL of BG11 (NO3-) supplemented with 5 µg / mL kanamycin. The microplates were incubated at 30°C under continuous light at an intensity of 45 μE·m−2·s−1. Optical density at 730 nm was measured hourly to monitor growth. At 24 hours, 25 µL concentrated NaCl stock in BG11 (NO3-) was added into culture for salt induction, and hourly data collection continued until 54 hours. The growth rates before and after salt induction were analyzed using an exponential growth model.

[0107] 1.7. High-throughput assay of biomass and sucrose productivities

[0108] Mutant colonies were maintained on agar plates and transferred to 96-well plates, each well containing 250 µL of BG11 (NO3-). Growth and salt induction were conducted following the optimized procedure above. Optical densities at inoculation (OD730nm, 0h), salt induction (OD730nm, 24h), and post incubation (OD730nm, 54h) were measured using a Molecular Devices SpectraMax® iD5 Multi-Mode Microplate Reader. Post incubation, cells were subjected to sucrose assay using the Eppendorf epMotion® 5073m liquid handling workstation.Attorney Docket no.10457-590PC0 Cyanobacterial cells are lysed with the addition of 25 µL of lysis solution (2% dodecyl trimethylammonium bromide (DTAB) in 0.4 M NaOH) per 50 µL sample, followed by neutralization with 0.4 M HCl after 5 minutes of shaking at 1,100 rpm. Post lysis, 50 µL of invertase (a glycoside hydrolase enzyme that breaks down sucrose into glucose and fructose) solution was added, incubated at 50°C for 1 hour, followed by the addition of 100 µL of Megazyme GOPOD reagent and a further 20 minute incubation at 50°C. OD510nm was then measured, and sucrose concentration was calculated against the standard curve ranging from 0.05 mg / mL to 0.1 mg / mL. Sucrose productivity is calculated by incorporating growth rate before (r1) and after (r2) salt induction. Given the exponential growth model, the following formula was used to calculate sucrose productivity:

[0109]

[0110] This method captures dynamic growth changes influenced by environmental conditions, facilitating a more accurate assessment of the mutants' sucrose productivity relative to the growth.

[0111] 1.8. Identification and validation of candidate strains from high-throughput screening

[0112] To identify potential candidate strains with increased sucrose productivities, the mSe0 strain was used to construct a model to estimate the relationship between sucrose productivity and biomass. To do this, mSe0 was randomly inoculated into 96-well plates across a spectrum of OD730 values. Sucrose and growth assays were performed as described above. The obtained data formed the training set, which was first grouped using k-mean clustering, followed by analyzing their relationships using a linear regression-based model. This model used log-transformed sucrose productivity as the response variable, and terminal OD730 as the independent variable:

[0113]

[0114] Sucrose-producing candidates were selected based on the highest 1% standardized residuals of sucrose productivities at different OD730nm. Additionally, mutants in the top 0.25 % of both terminal biomass (OD730nm, 54h) and biomass fold change (OD730nm, 54h / OD730nm, 0h) were selected as biomass-accumulating candidates and fast-growing candidates, respectively.

[0115] To identify elite mutants with statistical significance compared with the control strain mSe0, power analysis was used to establish an appropriate sample size, ensuring a power of 0.9 and an alpha threshold of 0.05. Assays for sucrose productivity, growth rate, and biomass accumulation were repeated following initial screening procedures. Statistical analyses were performed using block ANOVA, with 'block' representing different experimental batches.Attorney Docket no.10457-590PC0

[0116] 1.9. Correlation analysis between chlorosis and multiple variables

[0117] To assess the impact of chlorosis on sucrose production and growth experienced by some mutants during high-throughput screening, Pearson correlation analysis was performed. The chlorosis status of mutants was first determined using a classifier trained on a dataset including mSe0 and a subset of mSe3 cells. The variables include sucrose productivity, OD730nm, 54h, growth rate OD630nm, 54h, and phycobilin change rate post salt induction All predictors were standardized by their means and standard deviations to

[0118] 1.10. Genome extraction and sequencing

[0119] The cyanobacterial culture was grown in BG11 (NO3-) supplemented with 20 mM NaHCO3 and 5 µg / mL kanamycin. Cells were harvested for genome extraction when OD730nm reached between 0.5 and 0.6. The Promega Wizard® HMW DNA Extraction Kit was employed for genomic DNA (gDNA) extraction, using approximately 3 × 109cells. The extracted genomic DNA, with yields exceeding 1.2 µg, was utilized for PCR-free library preparation and subsequent genome sequencing by Novogene with an average of 1,000 × genome coverage based on 150 bp paired-end reads.

[0120] 1.11. Genetic variant calling and mutation analysis

[0121] To ensure the sequencing quality, all FASTQ files were inspected using FastQC v0.11.948. Low-quality bases in each FASTQ file were later trimmed using Trimmomatic v0.3949. Adapters and low-quality reads including leading and trailing bases below quality 3 or N bases were removed. A 4-base window was used to slide and scan all reads and cut reads if average quality was less than 15. At the end, remaining reads shorter than 36 bases were discarded. The trimmed reads were then mapped against the S. elongatus PCC 7942 reference genome from NCBI (Assembly: GCF_000012525.1) using Bowtie2 v2.2.5 with default parameters50. Variant calling for SNPs and indels was performed using samtools and bcftools (version 1.9)51. The process involved converting SAM files to BAM format, sorting the BAM files by genomic location, and converting sorted BAM files to text pileup output. Specifically, no anomalous read pairs were skipped, and both base and mapping quality thresholds were set to 0. In addition, several annotation information was added including allelic depth (AD), allelic depths on the forward strand (ADF), allelic depth on the reverse strand (ADR), number of high-quality bases (DP),Attorney Docket no.10457-590PC0 Phred-scaled strand bias P-value (SP). bcftools call was then used to obtain mutations. All alternative alleles present in the alignments even if they did not appear in any genotypes were collected, multiallelic caller was adopted due to the polyploidy of cyanobacteria. Genotype quality (GQ) was assessed for calling quality. The resultant VCF file underwent annotation with snpEff v5.2 using default settings, and annotated VCF files were tabularized using the snippy-vcf_to_tab function in the snippy package (v4.6.0)52. A customized R script was used to perform all allele frequency analysis.

[0122] 1.12. Mutation complementation

[0123] The psbA1 mutations were introduced in reverse primers and amplified with a universal forward primer for constructing all complementing mutant strains. The amplified psbA1 genes were purified and assembled using NEBuilder HiFi DNA Assembly Master Mix, and the constructs were transformed into S. elongatus PCC 7942 wild type. New colonies were initially grown on low-phosphate BG11 supplemented with 5 μg / mL chloramphenicol for two days to enable faster segregation, then transferred to standard BG11 plate supplemented with 5 μg / mL chloramphenicol. Segregation was verified by PCR amplification of the NSIII region. Transformants were cultured in 24-well plates for preculture, then transferred to 96-well plates containing BG11 (NO3-) with 20 mM NaHCO3with or without 150 mM NaCl. Sixteen replicates per mutant per condition were inoculated to ensure statistical robustness. Mutants were exposed to a light intensity of 110 μE m−2s−1(moderate light stress) and / or 150 mM NaCl to assess growth. OD730nm readings were taken at 0h, 6h, and either 18h or 30h depending on whether salt was added. For batch cultures, mutants were grown in multi-cultivators at 100 μE m−2s−1and 30°C, harvested at mid-log phase, and inoculated in triplicate into new media at a starting OD730nm of 0.05. Growth was monitored under different light conditions (100, 300, 1,500 μE m−2s−1) and salt concentrations (0, 150, 300 mM NaCl).

[0124] 1.13. Oxygen evolution and full light spectrum absorbance measurements

[0125] During mid-log phase, cell cultures were sampled for oxygen evolution using a Hansatech Oxytherm+ system. After a 10-minute dark incubation, oxygen evolution was measured for 7 minutes either at growing light intensities or saturating light intensity using the built-in LED panel, followed by the measurement of respiration immediately after termination of illumination. Oxygen evolution rates were normalized to biomass OD730nm. For absorbance measurements, biological triplicates with three technical replicates of 250 µL cell culture were analyzed in aAttorney Docket no.10457-590PC0 BioTek Synergy H1 plate reader by collecting full light spectrum absorbance from 300 nm to 700 nm at 2 nm intervals. The absorbance at 730 nm was used to normalize the pigment content.

[0126] 1.14 Pigment quantification

[0127] A previously published protocol was adopted with some modifications53. 1.5 mL of mid- log phase cyanobacterial cultures were collected by centrifugation at 15,000 rpm for 5 min. Cell pellet was resuspended in 1.5 mL 90 % (Vol / Vol) cold acetone by vortexing. Cells were transferred to 2mL Bead-Beating DuraTubes and subjected to 2-3 cycles of bead-beating for 45 seconds each, with cooling on ice between cylces. After cell lysis, samples were centrifuged again for 2 min at 15,000 rpm. Supernatant were aliquoted onto 96-well microplate for measurement of A665 and A720 on the plate reader. Chlorophyll a was calculated as follows:

[0128] . mutagenesis by controlling nitrogen sources

[0130] The overall experimental design features directed evolution using a hypermutator strain, followed by high-throughput screening in 96-well microplates to identify mutants with enhanced carbon fixation under salt stress. Experimental evolution with S. elongatus can be time-consuming due to its relatively slow doubling time, which approximates 10 hours under optimal growth conditions23.

[0131] Disrupting the mutS gene of the methyl-directed mismatch repair system (MMR) has been shown effective in inducing higher mutation rates in S. elongatus genome19. To enable accelerated evolution and facilitate downstream high-throughput screening, it was sought to construct a hypermutator strain that can easily switch between hypermutation and normal states to effectively induce mutagenesis and preserve mutations. To achieve this, a previously published method to construct a S. elongatus hypermutator strain mSe0 (mutS0::nirAp), was adopted by placing the mutS gene under an inducible promoter PnirA controlled byIn doing so, the hypermutation state is induced in cells grown in BG11 medium supplemented solely with ammonium, and mutations are maintained by switching to BG11 medium exclusively supplemented with nitrate (Fig. 2a).

[0132] To confirm the higher mutation rate of the mSe0 strain, a spot assay was first conducted by diluting an equal amount of mSe0 cells onto BG11 medium supplemented exclusively with either nitrate or ammonium. Higher mutation rates increase the probability of hitting lethal genes, resulting in smaller viable cell populations in ammonium medium compared with nitrate (Fig.2b).Attorney Docket no.10457-590PC0

[0133] RT-qPCR further confirmed that the mutS gene expression in the mSe0 strain was reduced in ammonium compared with nitrate, and was lower than that in wild-type S. elongatus under the ammonium condition (Fig. 2c).

[0134] Example 3. Mutagenesis and high-throughput screening of cyanobacterial mutants with increased biomass and / or sucrose productivity under salt stress

[0135] The hypermutator strain underwent three rounds of mutagenesis experiments under the light intensity of 45 μE·m−2·s−1in BG11 medium supplemented with increasing concentrations of NaCl (150 mM, 200 mM, and 250 mM). Each mutagenesis session was limited to four days, or approximately 8-10 cell generations, to carefully control mutation accumulation. This approach aims to generate potential Pareto-front genetic traits that enhance photosynthesis under salt stress without compromising normal growth performance (Fig. 3). After each round, mutants were grown in BG11 nitrate medium supplemented with NaCl and screened for enhanced carbon fixation.

[0136] To effectively identify cyanobacterial mutants with enhanced photosynthesis under salt stress, a high-throughput screening method was developed to isolate desired mutants. The most reliable indicator of improved photosynthesis is an increase in terminal biomass, resulting from compounded higher carbon fixation over the cultivation period. Thus, biomass accumulation was used as one indicator of improved photosynthesis during the screening phase.

[0137] In cyanobacteria, sucrose serves as a compatible solute for salt acclimation25. Sucrose is also the major photosynthesis end product in most plant leaves, with its synthesis closely linked to the rate of carbon fixation26. Therefore, sucrose accumulation was used as a second indicator for improved photosynthesis in S. elongatus mutants.

[0138] The first two rounds of mutagenesis, conducted under NaCl concentrations of 150 mM and 200 mM, served as preliminary screening steps to isolate potential candidate mutants. Sucrose productivity was utilized as the sole quantitative metric for these rounds, selecting those mutants with the highest sucrose productivities within the population as final candidates. These candidate strains from each round were further engineered to secrete sucrose by incorporating the Escherichia coli sucrose permease (cscB) gene, as evidenced previously that increased sucrose production is closely linked to higher photosynthetic efficiency in the sucrose-secreting S. elongatus27. From the initial round of mutagenesis, approximately 700 mutants (mSe1) were screened, identifying mSe1-1086 as the final candidate strain. This strain served as the progenitorAttorney Docket no.10457-590PC0 for the second round of mutagenesis under 200 mM NaCl, with approximately 1,200 mutants (mSe2) screened. Among these, the strain mSe2-842, exhibiting higher sucrose productivity compared with the sucrose-secreting S. elongatus control, was then selected as the starting strain for the subsequent round.

[0139] A larger-scale screening was conducted for the third round of mutants (mSe3) that underwent mutagenesis in BG11 supplemented with 250 mM NaCl. To increase the accuracy in isolating desired mutants, the inoculation and cultivation procedures were optimized to ensure that most mutants remained in the exponential growth phase during the high-throughput screening step. Further, a regression model was developed to quantitatively distinguish potential candidate mutants from controls based on sucrose productivity, using mSe0 as the baseline. This segmented linear model estimates the relationship between sucrose productivity and biomass for the control strain. Sucrose productivity of mSe3 mutants were then evaluated against this model to identify Sucrose-Producing Mutants (SPMs) with the highest 1% standardized residuals of sucrose productivities at different OD730nm.

[0140] During the screening process, a subset of cells exhibited chlorosis. To assess its impact on sucrose productivity, a correlation analysis between different variables and chlorosis was constructed. The variables include terminal biomass (OD730nm, 54h), terminal phycobilin level (OD630nm, 54h), sucrose productivity, growth rate post-salt induction (OD730nm, 54h / OD730nm, 24h), and phycobilin pigment change rate (OD630nm, 54h / OD630nm, 24h). Correlation analysis showed no significant link between chlorosis and sucrose productivity (score of 0.28), suggesting that chlorosis does not affect sucrose production.

[0141] For biomass accumulation, the terminal biomass (OD730nm, 54h) was used to determine potential Biomass-Accumulating Mutants (BAMs). Additionally, the biomass of a subset of mSe3 mutants was normalized to the initial inoculation density (OD730, 54h / OD730, 0h) to reflect their growth rates, with top candidates designated as Fast- Growing Mutants (FGMs). The inclusion of both terminal biomass and growth rate in our analysis is justified by the moderate correlation (score of 0.76) between the two, suggesting that each parameter captures distinct variables essential for identifying elite performers.

[0142] In total, over 10,000 mSe3 mutants were screened, the top 1% based on sucrose productivity selected as SPM candidates (Fig. 4). Additionally, the top 0.25% based on terminal biomass (OD730nm, 54h) were identified as BAM candidates (Fig. 4), and another top 0.25%Attorney Docket no.10457-590PC0 mutants based on biomass fold change (OD730nm, 54h / OD730nm, 0h) were selected as FGM candidates (Fig. 4 inset).

[0143] To validate the effectiveness of this models and identify elite mutants for either sucrose production or biomass accumulation, validation assays were conducted with sufficient replicates to achieve a statistical power of 90%. Using block ANOVA for comparison against the mSe0 control, six mutants were identified as the final elite BAM or FGM mutants with increased biomass accumulation, including mSe3-342, mSe3-470, mSe3-1291, mSe3-1804, mSe3-7265, mSe3-8345 (Fig. 5a). Additionally, two mutants, mSe3-7803 and mSe3-7976, were confirmed as elite Sucrose-Producing Mutants (SPMs) through block ANOVA (Fig. 5b). Importantly, these mutants exhibited similar photosynthetic rates to the S. elongatus wild type in batch cultures without stress, suggesting that potential mutations conferring salt tolerance did not compromise their normal growth performance. Interestingly, the mSe-342 strain, which was later discovered to exhibit a filamentous phenotype, failed to grow in the multi-cultivator, likely due to cell fragmentation caused by bubbling during growth.

[0144] Example 4. Mutations in BAM, FGM, and SPM elite strains

[0145] All eight elite strains along with S. elongatus wild type were sequenced with an average 1,000 × sequence coverage to identify potential beneficial mutations. As S. elongatus contains 3- 4 genome copies during active growth28, a 0.25 allele frequency threshold was used to identify potential beneficial mutations, excluding the rest as potential sequencing errors or deleterious mutations.

[0146] Genome sequencing revealed mutations in S. elongatus wild type accumulated during domestication compared with the reference genome, including 19 missense / indel mutations and 10 synonymous mutations with a wide spectrum of allele frequencies (AFs). These loci were excluded from mSe3 strain mutations when identified with similar AFs. In total, 99 unique mutations were identified among all mSe3 strains with AFs > 0.25, with an average of 8-20 mutations found in each genome (Fig. 6). Among these, 32 mutations are in intergenic regions and 67 mutations in coding sequences (CDS). The majority of the CDS mutations are either missense or indel mutations, with 10 being synonymous mutations (Fig. 6).

[0147] Among the identified mutations, four elite mSe3 mutants (mSe3-470, mSe3-7803, mSe3- 2287976, and mSe3-8345) contain several mutations in the psbA1 gene, encoding the core PSII D1 protein29(Fig. 6b). The D1 protein is subjected to continuous photodamage and must beAttorney Docket no.10457-590PC0 replaced every few minutes to ensure optimal photosynthesis in cyanobacteria and other oxygenic phototrophs30. Salt stress has been shown to inhibit transcription and translation of D1 protein during photodamage repair in Synechocystis sp. PCC 680321. It is thus likely that mutations in the D1 gene might have led to improved growth under salt stress in the elite strains. The psbA1 gene in S. elongatus encodes 360 amino acids, termed pre-D1 (pD1). During PSII assembly, the 16 amino acids at the carboxyl terminus are cleaved to form the mature D1 (mD1) by the protease CtpA31. Interestingly, all point mutations identified in the psbA1 gene result in amino acid changes at the carboxyl terminus of the pD1 protein, leading to missense mutations at three positions, i.e. L353F, I358N, and H359N, either individually or in combinations (Fig.7). In cyanobacteria, pD1 processing is a two-step proteolytic process with the first cleavage step at the carboxyl terminus of Ala352 followed by the final cleavage at Ala34432,33. A previous study in Synechocystis sp. PCC 6803 further showed that Asn359 is crucial in preventing photoinhibition34. It is likely that these mutations near the proposed cleavage site or at the end of pD1 have led to more efficient cleavage during D1 maturation, resulting in longer duration of photochemical quenching and higher overall carbon fixation.

[0148] Among other identified mutations, an in-frame conversion of the start codon ATG to TTG in the mutS gene was found in several mutants, including mSe3-342, mSe3-470, mSe3-1291, mSe3-1804, and mSe3-7803, whereas mSe3-7265 had several mutations in the mutS coding region. These mutations might be results of survival strategies related to hypermutation. Many of the other identified CDS mutations are found in either hypothetical genes or obscure in their roles for photosynthesis improvement (Fig. 6b). The efforts were focused on validating the D1 gene mutations in potentially supporting improved photosynthesis in S. elongatus.

[0149] Example 5. Validating pD1 mutations toward tolerance to salt and light stress

[0150] High light results in the formation of reactive oxygen species and D1 damage, which is exacerbated by salt stress in cyanobacteria21. To test whether mutations in the psbA1 gene confer beneficial traits for stress tolerance to salt and possibly light as well, an additional copy of the psbA1 gene with either single mutations or their identified combinations was introduced into the neutral site III (NSIII) of the wild type S. elongatus genome, including psbA1L353F, psbA1I358N, psbA1H359N, psbA1T354P+I358N+H359N, and psbA1L353F+T354A+I358N. Mutations at the position 354, specifically T354P / A were not found in individual mutants but were found in lower than 0.25 frequencies together with the other mutations. Thus, the T354P / A mutations were included in theAttorney Docket no.10457-590PC0 validation experiment. A wild type psbA1 gene copy was similarly introduced to the genome to serve as the control (psbA1WT). A mixture of native and heterologous pD1s likely co-exists and is competitively recruited for PSII repair (Fig. 8).

[0151] Growth dynamics of these engineered D1 strains was tested under salt stress (150 mM NaCl) and elevated light conditions in both 96-well cell cultures (110 μE·m−2·s−1) and large batch cultures in multi-cultivators (300 μE·m−2·s−1). The pD1 mutations of L353F, I358N, H359N, and L353F+T354A+I358N all confer tolerance to salt stress and combined salt and light stress compared with the control (Fig.9). Interestingly, pD1 mutations of I358N and H359N do not seem to confer significant light tolerance on their own, suggesting salt tolerance as the dominant effect for their tolerance under combined salt and light stresses (Fig. 9). In conclusion, L353F, I358N, and H359N in the pD1 protein confer tolerance to both salt and combined salt and light stresses in S. elongatus.

[0152] Example 6. L353F mutation of pD1 improves photosynthesis under elevated salt and light stress

[0153] To understand how photosynthesis is impacted by these pD1 mutations under salt and light stress in S. elongatus, the L353F mutation was used as an example to test for two considerations. Firstly, the mutation occurs immediately after the proposed first-stage cleavage site for pD1 carboxyl terminus32(Fig. 7), thereby could be involved in improved cleavage kinetics during D1 maturation. Secondly, the H359N mutation has been previously reported, showing its supporting role to prevent photoinhibition in Synechocystis sp. PCC 680334. This suggests that asparagine near the end of pD1 could be important for PSII repair, as the I358N mutation also confers resistance to salt stress in our case (Fig. 9).

[0154] Both psbA1WTand psbA1L353Fstrains were grown under the elevated light intensity of 300 μE·m−2·s−1with three different NaCl levels (0 mM, 150 mM, and 300 mM) in multi-cultivators. The psbA1L353Fmutant exhibited similar growth to the psbA1WTstrain under 0 mM NaCl but showed increased growth under both 150 mM and 300 mM salt stress at the elevated light intensity (Fig. 10a). Full light spectrum scanning further revealed both increased chlorophyll (440 nm and 680 nm) and phycobilin (630 nm) contents in the psbA1L353Fmutant relative to the control (Fig. 10b), suggesting enhanced photosynthetic efficiencies under the combined salt and moderate light stress.Attorney Docket no.10457-590PC0

[0155] The increase in chlorophyll was not as robust when light alone was used as the stress factor at three different light intensities (100, 300, and 1,500 μE·m−2·s−1) , indicating that L353F confers a trait beneficial for salt tolerance, with an added advantage under moderate light stress. Oxygen evolution rate measured at growing light intensities further confirms the increased photosynthetic efficiency of the psbA1L353Fmutant compared with the control under salt stress (Fig. 10c). When measured with saturating light intensity, the maximum photosynthetic efficiency of the psbA1L353Fmutant is also higher than the control under 150 mM NaCl, but this difference is diminished under the higher salt level of 300 mM (Fig. 10d). Taken together, these results demonstrate that L353F mutation endows both increased photosynthetic capacity and kinetics under salt and moderate light stress in S. elongatus, exemplifying a Pareto-front genetic trait for enhancing photosynthesis under sublethal stress conditions.

[0156] Example 7. Engineering alternative D1 proteins in plants for photosynthesis improvement

[0157] The present invention relates to engineered constructs and methods for expressing cyanobacterial genes in plant systems to facilitate chloroplast-targeted functional studies and bioengineering applications. Specifically, the invention involves the heterologous expression of the psbA1 gene from Synechococcus elongatus PCC 7942, encoding the D1 protein of Photosystem II, in Arabidopsis thaliana. In one embodiment, psbA1 is expressed alone; in another embodiment, psbA1 is co-expressed with the cyanobacterial ctpA gene from the same organism. .For chloroplast localization, the psbA1 coding region was fused at its N-terminus to a chloroplast transit peptide derived from the Arabidopsis thaliana RuBisCO small subunit. Expression of this chimeric gene was driven by the enhanced Cauliflower Mosaic Virus 35S promoter, with transcriptional termination achieved using the CaMV polyadenylation signal. The construct was inserted into a binary vector containing selectable markers for kanamycin and hygromycin resistance.

[0158] In co-expression studies, the ctpA gene from Synechococcus elongatus was engineered with an N-terminal fusion to the chloroplast signal peptide of Arabidopsis CtpA, enabling chloroplast targeting of the heterologous protease. The expression of ctpA was driven by the Arabidopsis UBQ10 promoter and terminated using an HSP-derived transcriptional terminator. This construct was introduced on a separate binary vector containing kanamycin and bialaphos resistance genes.Attorney Docket no.10457-590PC0

[0159] Both single (psbA1 alone) and dual (psbA1 + ctpA) transformation approaches were performed in Arabidopsis thaliana using Agrobacterium tumefaciens-mediated floral dip. Transgenic lines were selected on media containing appropriate antibiotics corresponding to the selectable markers present in each construct. Molecular analyses were conducted to confirm transgene integration. The comparative expression systems allow assessment of the requirement for CtpA-mediated processing of the D1 protein in plant chloroplasts and enable photosynthesis optimization in higher plants.

[0160] 7.1. Genetic transformation of pD1 genes into Arabidopsis

[0161] To study how D1 variant affects Arabidopsis growth, different transgenic lines in Arabidopsis are generated by overexpressing cyanobacterial pD1 gene alone, pD1 along with native D1, and control null segregant lines.

[0162] The plasmid for introducing pD1 gene variants into Arabidopsis is assembled by Gibson Assembly. The plasmid is cloned in E. coli DH5α cells, followed by transformation into the Agrobacterium tumefaciens GV3101 strain. The Agrobacterium-mediated transformation is done using the Floral Dip method. The transgenic (T1) lines are regenerated from the seeds through plating on the selection medium. The transgenic lines are grown in trays in the growth chamber until two leaf growth, followed by growth in pots. The Arabidopsis wild type and a null segregant plant are used as control plants. The expression of D1 protein is confirmed by SDS-PAGE and immunoblot. The final D1-expressing transgenic lines are selected and further used for analysis under different conditions. See FIGs 13-16.

[0163] 7.2. Evaluation of growth improvement in transgenic lines

[0164] After obtaining homozygous transgenic lines, growth is assessed under different stress conditions to evaluate the impact of cyanobacterial D1 protein on the Arabidopsis growth. High light conditions (500 μmol m−2 s−1) and / or salt (80 mM NaCl) are applied under day night cycle (12h / 12h) with L / D temperatures of 23 / 18°C. The transgenic lines and control lines are assessed for their growth by measuring biomass using the whole rosette of individual plants. Seed yield and total shoot weight are measured upon complete growth after several weeks.

[0165] 7.3. Biochemistry analysis of pD1 from transgenic lines

[0166] Upon completion of the growth experiments, transgenic lines with improved growth are selected for biochemistry analyses to understand pD1 roles. The in vitro enzymatic cleavage assay is conducted to measure turnover rates of cyanobacterial pD1 and plant native pD1. ThylakoidAttorney Docket no.10457-590PC0 enriched PSII is used as substrates for the kinetics studies. To initiate the enzymatic assay, recombinant Arabidopsis CtpA gene is purified from E. coli BL21. The proteolytic reaction is conducted at 21°C under different light intensities (25, 100, 250 and 500 μmol m−2 s−1)for 0, 5, and 10 min followed by immunoblotting to compare the proteolytic cleavage activity of CtpA on cyanobacterial pD1 and plant-native pD1 by quantifying pD1 and mD1 protein levels after the enzymatic reaction.

[0167] 7.4. Measurement of photosynthetic parameters in transgenic lines

[0168] Enhanced turnover of the D1 protein is likely lead to reduced photoinhibition and enhanced photosynthesis in transgenic lines under high light conditions. To test this, a series of photochemistry measurements are conducted to gauge how PSII activities are affected in the transgenic lines. Leaf samples are collected from soil-grown plants under standard illumination (100 μmol m-2s-1), followed by a 3-hour water soak under an irradiance of 15 µE. The leaf samples are then exposed to inhibitory irradiance (1800 μmol m-2s-1), followed by a 20-minute dark adaptation period to open PSII reaction centers. Photosynthesis efficiency parameters including the maximum PSII activity (Fv / Fm), and effective PSII activity (ΦPSII), and the non-photochemical quenching (NPQ) are recorded to understand whether there are any changes in photosynthetic efficiency in these transgenic lines.

[0169] Incorporating the cyanobacterial pD1 protein variant into plants may not have any obvious effect under optimal growth conditions. However, improved growth and biomass accumulation (seed yield and total shoot dry weight) is demonstrated along with increased PSII activity in transgenic lines under fluctuating environmental conditions. In addition, the enzyme kinetics data provides strong support on the contribution of pD1 processing in enhancing photosynthesis and plant growth. The data collectively demonstrate the effectiveness of engineering beneficial genetic traits from cyanobacteria to improve plant growth. These data will provide important insights for engineering D1 and other beneficial genetic traits in crop plants for increased biomass yield and food production.

[0170] 7.5. D1 mutation L353F promoting root development

[0171] The psbA1 gene from the cyanobacterium Synechococcus elongatus PCC 7942 was introduced into Arabidopsis thaliana to evaluate its impact on plant photosynthesis and growth. Both a wild-type cyanobacterial copy (WT) and a mutated D1 variant carrying the L353F substitution were integrated into the nuclear genome of Arabidopsis. See Fig. 17. It shows psbA1Attorney Docket no.10457-590PC0 gene from the cyanobacterium Synechococcus elongatus PCC 7942 was introduced into Arabidopsis thaliana to evaluate its impact on plant photosynthesis and growth. Both a wild-type cyanobacterial copy (psbA1) and a mutated D1 variant carrying the L353F substitution (psbA1L353F) were integrated into the nuclear genome of Arabidopsis. The images below represent two homozygous T3 lines of each transformant. The current results show that the D1 mutation L353F derived from cyanobacteria promotes root development. Specifically, the psbA1L353Flines exhibited longer roots and more robust root hairs. In addition, the transformants grow better under salt conditions as well compared to Col-0 control.

[0172] Example 8: Pathogen Resistance

[0173] Also disclosed herein is data showing that the modification of plants to express or overexpress at least one variant of pre-D1 (pD1) protein increases resistance to plant pathogen infection. This involves transfecting cells of a plant with at least one variant of psbA1 gene encoding the variant pD1 protein, and wherein the variant psbA1 gene is a mutated psbA1 gene having at least one point mutation in a wild type psbA1 gene from cyanobacterium, Synechococcus elongates, and / or optionally from S. elongates PCC 7942 strain into the plant to increases resistance of the plant to infection by plant pathogens. See FIG. 18.

[0174] Reference to “maintaining plant growth” as used herein means that the growth of a plant or parts thereof (such as roots and shoots) which has been exposed to, infected with or which is susceptible to a plant pathogen, is the same or at least 50-70% the same as compared to a control plant or parts thereof which has not been exposed to, infected with or which is not susceptible to a pathogen, or as compared to a predetermined standard. Alternatively, “maintenance of plant growth” can be determined by recording differences in biomass between a plant or parts thereof which has been exposed to, is infected with or which is susceptible to a pathogen, as compared to the biomass of a control plant or parts thereof which has not been exposed to, infected with or which is not susceptible to a pathogen, or as compared to a predetermined standard.

[0175] The term “plant biomass” as used herein means biological material derived from a living, or recently living plant. Methods for measuring plant biomass may be employed, for example, by taking samples of plant tissues (which may be roots, shoots, leaves, heads or grains) and drying at +60° C. until the mass no longer changes upon further drying. Without being limited by anyone theory or mode of action, plant biomass is generally taken on replicate samples (minimum 3) and presented as the mean dry mass+ / −standard error of the mean. The dry mass can be usedAttorney Docket no.10457-590PC0 individually as “tissue biomass” or summed to give “above ground biomass”, “below ground biomass” or “total biomass”.

[0176] In the context of the present specification “maintaining the yield” of a plant crop which has been exposed to, infected with or which is susceptible to a plant pathogen, is to be understood to refer to the same or at least 50-70% the same as compared to a control plant or parts thereof which has not been exposed to, infected with or which is not susceptible to a pathogen, or as compared to a predetermined standard with respect to any measure of output (typically agricultural or horticultural output) from the crop including, by way of example only, maintaining the biomass of the plant or one or more plant parts (such as edible plant parts), or maintaining plant growth, such as for example, maintaining the number, size, volume, viability or quality of one or more plant parts such as fruit, vegetable, tuber, seed, nut, flower, stalk, stem, leaf or any other plant part that has economic value as compared to a control.

[0177] As used herein the terms “treating”, “treatment”, “preventing” and “prevention” refer to any and all uses which remedy a disease condition or symptoms caused by or associated with a pathogen infection, prevent the establishment of a disease caused by or associated with a pathogen infection, or otherwise prevent, hinder, retard, or reverse the progression of a disease or other undesirable symptoms caused by or associated with a pathogen infection in any way whatsoever. Thus the terms “treating” and “preventing” and the like are to be considered in their broadest context. For example, treatment does not necessarily imply that the plant is treated until total recovery. In conditions which display or are characterized by multiple symptoms, the treatment or prevention need not necessarily remedy, prevent, hinder, retard, or reverse all of said symptoms, but may prevent, hinder, retard, or reverse one or more of said symptoms. In the context of some plant diseases caused by or associated with a pathogen infection, methods of the present invention involve “treating” the disease in terms of reducing or ameliorating the occurrence of a highly undesirable event associated with the disease or an irreversible outcome of the progression of the disease but may not of itself prevent the initial occurrence of the event or outcome. Accordingly, treatment includes amelioration of the symptoms of a particular disease or preventing or otherwise reducing the risk of developing a particular disease.

[0178] As used herein reference to “reducing the susceptibility” of a plant to a pathogenic infection should be understood to refer to the process of protecting the plant from infection by a plant pathogen, including protecting a healthy plant free from disease. It should be understood that aAttorney Docket no.10457-590PC0 reduction in susceptibility does not necessarily imply that a plant will no longer develop a pathogenic infection. Rather a reduction in susceptibility means that the likelihood that a plant will develop a disease caused by or associated with a pathogen infection is less than the likelihood that a plant which has not undergone treatment will develop a disease caused by or associated with a pathogen infection.

[0179] The present disclosure provides an effective, simple, economical means for controlling disease caused by, or associated with a pathogen in plants and of maintaining plant growth and / or crop yield by reducing plant pathogen susceptibility and enabling plants to survive and grow even in disease prone environments which has the particular benefit of reduced or minimal harm upon ecosystems or the environment.

[0180] Current management strategies have limited effectiveness against plant fungal pathogens including, for example, those from the genus Fusarium, Gaeumannomyces, Rhizoctonia, Pythium, Sclerotinia, Alternaria, and Blumeria. As noted elsewhere, the inventors have surprisingly found that a composition comprising Streptomyces isolates MH71 and MH243 applied to plants, including plant seeds, exhibited a high biofungicide efficacy against a broad range of plant fungal pathogens.

[0181] In a particular embodiment of the disclosure the plant pathogen is a fungal pathogen. Illustrative examples of fungal pathogens contemplated by the present disclosure include, but are by no means limited to, those pathogens belonging to the Fusarium spp. (e.g. causal agents of Fusarium wilt disease, Fusarium crown rot disease and Fusarium head blight), Gaeumannomyces graminis (e.g. causal agent of take-all root rot), Leptosphaeria maculans (e.g. causative agent of blackleg), Sclerotinia sclerotiorum (e.g. causal agents of Sclerotinia stem rot, also known as white mould / mold, cottony rot; watery soft rot; and blossom blight), Alternaria spp. (e.g. A. brassicicola blight), Ustilago spp. (e.g. the causal agents of smut), Rhizoctonia spp. (e.g. causal agents of root and hypocotyl rot and of barepatch), Pythium irregulare (e.g. causal agent of Pythium root rot), Thielaviopsis spp. (e.g. causal agents of canker rot, black root rot, Thielaviopsis root rot), Verticillium spp., Magnaporthe grisea (e.g. causal agent of rice blast), Phakospora pachyrhizi (e.g. causal agent of soybean rust), Puccinia spp. (e.g. causal agents of severe rusts of virtually all cereal grains and cultivated grasses), Blumeria graminis (powdery mildew of wheat), Erysiphe spp. (e.g. Erysiphe necator causal agent of powdery mildew of grapes), Botrytis cinerea (causal agent ofAttorney Docket no.10457-590PC0 botrytis bunch rot and grey mould / gray mold) and Armillaria spp. (e.g. the so-called honey fungus species, which are virulent pathogens of trees and produce edible mushrooms).

[0182] In another embodiment, illustrative examples of bacterial plant pathogens for which the methods herein reduce susceptibility include, but are by no means limited to those pathogens belonging to the genus selected from Xanthomonas (e.g. Xanthomonas oryzae pv. oryzae; Xanthomonas campestris pathovars; Xanthomonas translucens pathovars; and Xanthomonas axonopodis pathovars), Pseudomonas (e.g. Pseudomonas syringae pathovars; and Pseudomonas fuscovaginae), Ralstonia (e.g. Ralstonia solanacearum), Agrobacterium (e.g. Agrobacterium tumefaciens), Erwinia (e.g. Erwinia amylovora—also known as fireblight), Xylella (e.g. Xylella fastidiosa), Dickeya (e.g. dadantii and solani), Pectobacterium (e.g. Pectobacterium carotovorum and Pectobacterium atrosepticum), Clavibacter (e.g. Clavibacter michiganensis (ring rot) and Clavibacter sepedonicus) and Candidatus (e.g. Candidatus Liberibacter asiaticus).

[0183] The potential to reduce the susceptibility of a plant to a plant pathogen infection, has significant potential commercial and environmental benefits including in broad acre crop production, horticulture for food or fibre, ornamentals, native ecosystem establishment and rehabilitation, plantation forestry, mine site restoration, landscaping, agriculture, plant propagation in nurseries, and other related industries. Therefore, the present disclosure may be applied to any plant species. These benefits may manifest in plants grown under a variety of conditions (including, for example, field, glasshouse, container or vat grown).

[0184] To assess basal resistance to Psm, two leaves from each 4-week-old plant were infiltrated with a Psm suspension (OD600 = 0.0003). Bacterial populations were measured 3 dpi to evaluate pathogen presence. The results show that all transgenic lines have higher basal resistance compared to the Col-0 wild type. In particular, the psbA1L353 -4 line showed the highest resistance to the pathogen Psm. See Fig. 18.

[0185] Example 9: Relevant Sequences

[0186] SEQ ID NOs: 1 and 2 are directed to wild type PsbA1 Synechococcus elongatus PCC 7942 and SEQ ID NOs: 3-10 relate to mutations of either SEQ ID NOs: 1 (nucleic acid sequence) or 2 (amino acid sequence). SEQ ID NO: 11 relates to the amino acid sequence of CtpA of Synechococcus elongatus PCC 7942.Attorney Docket no.10457-590PC0 >psbA1_wild type NC_007604.1:413873-414955 Synechococcus elongatus PCC 7942 = FACHB- 805, complete sequence (SEQ ID NO: 1) ATGACCAGCATTCTTCGCGAGCAACGCCGCGATAACGTTTGGGATCGGTTTTGTGAGT GGGTAACCAGCACCGACAACCGCATCTACGTGGGTTGGTTCGGCGTGCTGATGATCC CCACTCTGCTGACCGCCACCATCTGCTTCATCGTTGCGTTCATTGCAGCCCCTCCCGT CGACATCGACGGCATCCGTGAGCCCGTTGCCGGCTCTCTCATGTATGGCAACAACATC ATTTCCGGCGCTGTTGTTCCTTCCAGCAACGCCATCGGCCTGCATTTCTATCCGATTTG GGAAGCCGCTAGCCTCGACGAGTGGCTGTACAACGGTGGTCCTTACCAATTAGTGGT CTTCCACTTCTTGCTGGGTATCAGCTGCTACATGGGTCGTCAATGGGAGCTGTCGTAC CGCCTCGGTATGCGCCCTTGGATCTGTGTTGCATACAGTGCTCCACTCTCGGCTGCTTT TGCAGTGTTTCTGATCTACCCGATCGGCCAAGGTTCGTTCTCGGACGGCATGCCCCTG GGTATCAGCGGCACCTTCAACTTCATGTTCGTGTTCCAAGCAGAGCACAACATTTTGA TGCACCCCTTCCACATGCTGGGTGTGGCTGGTGTGTTCGGTGGTTCGCTGTTCTCGGC AATGCACGGTTCGTTGGTGACCAGCTCGCTGGTGCGTGAGACGACCGAGACCGAGA GCCAAAACTACGGCTACAAATTTGGTCAAGAGGAAGAGACCTACAACATCGTGGCAG CCCACGGTTACTTCGGTCGCTTGATCTTCCAATACGCATCGTTCAACAACAGCCGTTC GCTGCACTTCTTCCTGGGTGCATGGCCGGTCGTGGGCATCTGGTTTACCTCCATGGGC ATCAGCACCATGGCGTTCAACCTGAATGGTTTCAACTTCAACCAGTCGGTTTTGGATA GCCAAGGCAAAGTGATCAACACTTGGGCAGATGTGTTGAACCGTGCCAACTTGGGCA TGGAAGTGATGCACGAGCGTAATGCTCACAACTTCCCGCTCGACTTGGCAGCAGGCG AAGCGACCCCGGTCGCTTTGACTGCGCCTTCAATTCACGGTTAA >P04996 PSBA1_SYNE7 Photosystem II protein D11 OS=Synechococcus elongatus (strain ATCC 33912 / PCC 7942 / FACHB-805) OX=1140 GN=psbA1 PE=1 SV=1 (SEQ ID NO: 2) MTSILREQRRDNVWDRFCEWVTSTDNRIYVGWFGVLMIPTLLTATICFIVAFIAAPPVDID GIREPVAGSLMYGNNIISGAVVPSSNAIGLHFYPIWEAASLDEWLYNGGPYQLVVFHFLL GISCYMGRQWELSYRLGMRPWICVAYSAPLSAAFAVFLIYPIGQGSFSDGMPLGISGTFNF MFVFQAEHNILMHPFHMLGVAGVFGGSLFSAMHGSLVTSSLVRETTETESQNYGYKFGQ EEETYNIVAAHGYFGRLIFQYASFNNSRSLHFFLGAWPVVGIWFTSMGISTMAFNLNGFN FNQSVLDSQGKVINTWADVLNRANLGMEVMHERNAHNFPLDLAAGEATPVALTAPSIH GAttorney Docket no.10457-590PC0 >OE6-I358N (SEQ ID NO: 3) ATGACCAGCATTCTTCGCGAGCAACGCCGCGATAACGTTTGGGATCGGTTTTGTGAGT GGGTAACCAGCACCGACAACCGCATCTACGTGGGTTGGTTCGGCGTGCTGATGATCC CCACTCTGCTGACCGCCACCATCTGCTTCATCGTTGCGTTCATTGCAGCCCCTCCCGT CGACATCGACGGCATCCGTGAGCCCGTTGCCGGCTCTCTCATGTATGGCAACAACATC ATTTCCGGCGCTGTTGTTCCTTCCAGCAACGCCATCGGCCTGCATTTCTATCCGATTTG GGAAGCCGCTAGCCTCGACGAGTGGCTGTACAACGGTGGTCCTTACCAATTAGTGGT CTTCCACTTCTTGCTGGGTATCAGCTGCTACATGGGTCGTCAATGGGAGCTGTCGTAC CGCCTCGGTATGCGCCCTTGGATCTGTGTTGCATACAGTGCTCCACTCTCGGCTGCTTT TGCAGTGTTTCTGATCTACCCGATCGGCCAAGGTTCGTTCTCGGACGGCATGCCCCTG GGTATCAGCGGCACCTTCAACTTCATGTTCGTGTTCCAAGCAGAGCACAACATTTTGA TGCACCCCTTCCACATGCTGGGTGTGGCTGGTGTGTTCGGTGGTTCGCTGTTCTCGGC AATGCACGGTTCGTTGGTGACCAGCTCGCTGGTGCGTGAGACGACCGAGACCGAGA GCCAAAACTACGGCTACAAATTTGGTCAAGAGGAAGAGACCTACAACATCGTGGCAG CCCACGGTTACTTCGGTCGCTTGATCTTCCAATACGCATCGTTCAACAACAGCCGTTC GCTGCACTTCTTCCTGGGTGCATGGCCGGTCGTGGGCATCTGGTTTACCTCCATGGGC ATCAGCACCATGGCGTTCAACCTGAATGGTTTCAACTTCAACCAGTCGGTTTTGGATA GCCAAGGCAAAGTGATCAACACTTGGGCAGATGTGTTGAACCGTGCCAACTTGGGCA TGGAAGTGATGCACGAGCGTAATGCTCACAACTTCCCGCTCGACTTGGCAGCAGGCG AAGCGACCCCGGTCGCTTTGACTGCGCCTTCAAaTCACGGTTAA >OE6-I358N (SEQ ID NO: 4) MTSILREQRRDNVWDRFCEWVTSTDNRIYVGWFGVLMIPTLLTATICFIVAFIAAPPVDID GIREPVAGSLMYGNNIISGAVVPSSNAIGLHFYPIWEAASLDEWLYNGGPYQLVVFHFLL GISCYMGRQWELSYRLGMRPWICVAYSAPLSAAFAVFLIYPIGQGSFSDGMPLGISGTFNF MFVFQAEHNILMHPFHMLGVAGVFGGSLFSAMHGSLVTSSLVRETTETESQNYGYKFGQ EEETYNIVAAHGYFGRLIFQYASFNNSRSLHFFLGAWPVVGIWFTSMGISTMAFNLNGFN FNQSVLDSQGKVINTWADVLNRANLGMEVMHERNAHNFPLDLAAGEATPVALTAPSNH G >OE7-T354P (SEQ ID NO: 5)Attorney Docket no.10457-590PC0 ATGACCAGCATTCTTCGCGAGCAACGCCGCGATAACGTTTGGGATCGGTTTTGTGAGT GGGTAACCAGCACCGACAACCGCATCTACGTGGGTTGGTTCGGCGTGCTGATGATCC CCACTCTGCTGACCGCCACCATCTGCTTCATCGTTGCGTTCATTGCAGCCCCTCCCGT CGACATCGACGGCATCCGTGAGCCCGTTGCCGGCTCTCTCATGTATGGCAACAACATC ATTTCCGGCGCTGTTGTTCCTTCCAGCAACGCCATCGGCCTGCATTTCTATCCGATTTG GGAAGCCGCTAGCCTCGACGAGTGGCTGTACAACGGTGGTCCTTACCAATTAGTGGT CTTCCACTTCTTGCTGGGTATCAGCTGCTACATGGGTCGTCAATGGGAGCTGTCGTAC CGCCTCGGTATGCGCCCTTGGATCTGTGTTGCATACAGTGCTCCACTCTCGGCTGCTTT TGCAGTGTTTCTGATCTACCCGATCGGCCAAGGTTCGTTCTCGGACGGCATGCCCCTG GGTATCAGCGGCACCTTCAACTTCATGTTCGTGTTCCAAGCAGAGCACAACATTTTGA TGCACCCCTTCCACATGCTGGGTGTGGCTGGTGTGTTCGGTGGTTCGCTGTTCTCGGC AATGCACGGTTCGTTGGTGACCAGCTCGCTGGTGCGTGAGACGACCGAGACCGAGA GCCAAAACTACGGCTACAAATTTGGTCAAGAGGAAGAGACCTACAACATCGTGGCAG CCCACGGTTACTTCGGTCGCTTGATCTTCCAATACGCATCGTTCAACAACAGCCGTTC GCTGCACTTCTTCCTGGGTGCATGGCCGGTCGTGGGCATCTGGTTTACCTCCATGGGC ATCAGCACCATGGCGTTCAACCTGAATGGTTTCAACTTCAACCAGTCGGTTTTGGATA GCCAAGGCAAAGTGATCAACACTTGGGCAGATGTGTTGAACCGTGCCAACTTGGGCA TGGAAGTGATGCACGAGCGTAATGCTCACAACTTCCCGCTCGACTTGGCAGCAGGCG AAGCGACCCCGGTCGCTTTGcCTGCGCCTTCAATTCACGGTTAA >OE7-T354P (SEQ ID NO: 6) MTSILREQRRDNVWDRFCEWVTSTDNRIYVGWFGVLMIPTLLTATICFIVAFIAAPPVDID GIREPVAGSLMYGNNIISGAVVPSSNAIGLHFYPIWEAASLDEWLYNGGPYQLVVFHFLL GISCYMGRQWELSYRLGMRPWICVAYSAPLSAAFAVFLIYPIGQGSFSDGMPLGISGTFNF MFVFQAEHNILMHPFHMLGVAGVFGGSLFSAMHGSLVTSSLVRETTETESQNYGYKFGQ EEETYNIVAAHGYFGRLIFQYASFNNSRSLHFFLGAWPVVGIWFTSMGISTMAFNLNGFN FNQSVLDSQGKVINTWADVLNRANLGMEVMHERNAHNFPLDLAAGEATPVALPAPSIH G >OE8-H359N (SEQ ID NO: 7)Attorney Docket no.10457-590PC0 ATGACCAGCATTCTTCGCGAGCAACGCCGCGATAACGTTTGGGATCGGTTTTGTGAGT GGGTAACCAGCACCGACAACCGCATCTACGTGGGTTGGTTCGGCGTGCTGATGATCC CCACTCTGCTGACCGCCACCATCTGCTTCATCGTTGCGTTCATTGCAGCCCCTCCCGT CGACATCGACGGCATCCGTGAGCCCGTTGCCGGCTCTCTCATGTATGGCAACAACATC ATTTCCGGCGCTGTTGTTCCTTCCAGCAACGCCATCGGCCTGCATTTCTATCCGATTTG GGAAGCCGCTAGCCTCGACGAGTGGCTGTACAACGGTGGTCCTTACCAATTAGTGGT CTTCCACTTCTTGCTGGGTATCAGCTGCTACATGGGTCGTCAATGGGAGCTGTCGTAC CGCCTCGGTATGCGCCCTTGGATCTGTGTTGCATACAGTGCTCCACTCTCGGCTGCTTT TGCAGTGTTTCTGATCTACCCGATCGGCCAAGGTTCGTTCTCGGACGGCATGCCCCTG GGTATCAGCGGCACCTTCAACTTCATGTTCGTGTTCCAAGCAGAGCACAACATTTTGA TGCACCCCTTCCACATGCTGGGTGTGGCTGGTGTGTTCGGTGGTTCGCTGTTCTCGGC AATGCACGGTTCGTTGGTGACCAGCTCGCTGGTGCGTGAGACGACCGAGACCGAGA GCCAAAACTACGGCTACAAATTTGGTCAAGAGGAAGAGACCTACAACATCGTGGCAG CCCACGGTTACTTCGGTCGCTTGATCTTCCAATACGCATCGTTCAACAACAGCCGTTC GCTGCACTTCTTCCTGGGTGCATGGCCGGTCGTGGGCATCTGGTTTACCTCCATGGGC ATCAGCACCATGGCGTTCAACCTGAATGGTTTCAACTTCAACCAGTCGGTTTTGGATA GCCAAGGCAAAGTGATCAACACTTGGGCAGATGTGTTGAACCGTGCCAACTTGGGCA TGGAAGTGATGCACGAGCGTAATGCTCACAACTTCCCGCTCGACTTGGCAGCAGGCG AAGCGACCCCGGTCGCTTTGACTGCGCCTTCAATTaACGGTTAA >OE8-H359N (SEQ ID NO: 8) MTSILREQRRDNVWDRFCEWVTSTDNRIYVGWFGVLMIPTLLTATICFIVAFIAAPPVDID GIREPVAGSLMYGNNIISGAVVPSSNAIGLHFYPIWEAASLDEWLYNGGPYQLVVFHFLL GISCYMGRQWELSYRLGMRPWICVAYSAPLSAAFAVFLIYPIGQGSFSDGMPLGISGTFNF MFVFQAEHNILMHPFHMLGVAGVFGGSLFSAMHGSLVTSSLVRETTETESQNYGYKFGQ EEETYNIVAAHGYFGRLIFQYASFNNSRSLHFFLGAWPVVGIWFTSMGISTMAFNLNGFN FNQSVLDSQGKVINTWADVLNRANLGMEVMHERNAHNFPLDLAAGEATPVALTAPSIN G >OE9-L353F (SEQ ID NO: 9)Attorney Docket no.10457-590PC0 ATGACCAGCATTCTTCGCGAGCAACGCCGCGATAACGTTTGGGATCGGTTTTGTGAGT GGGTAACCAGCACCGACAACCGCATCTACGTGGGTTGGTTCGGCGTGCTGATGATCC CCACTCTGCTGACCGCCACCATCTGCTTCATCGTTGCGTTCATTGCAGCCCCTCCCGT CGACATCGACGGCATCCGTGAGCCCGTTGCCGGCTCTCTCATGTATGGCAACAACATC ATTTCCGGCGCTGTTGTTCCTTCCAGCAACGCCATCGGCCTGCATTTCTATCCGATTTG GGAAGCCGCTAGCCTCGACGAGTGGCTGTACAACGGTGGTCCTTACCAATTAGTGGT CTTCCACTTCTTGCTGGGTATCAGCTGCTACATGGGTCGTCAATGGGAGCTGTCGTAC CGCCTCGGTATGCGCCCTTGGATCTGTGTTGCATACAGTGCTCCACTCTCGGCTGCTTT TGCAGTGTTTCTGATCTACCCGATCGGCCAAGGTTCGTTCTCGGACGGCATGCCCCTG GGTATCAGCGGCACCTTCAACTTCATGTTCGTGTTCCAAGCAGAGCACAACATTTTGA TGCACCCCTTCCACATGCTGGGTGTGGCTGGTGTGTTCGGTGGTTCGCTGTTCTCGGC AATGCACGGTTCGTTGGTGACCAGCTCGCTGGTGCGTGAGACGACCGAGACCGAGA GCCAAAACTACGGCTACAAATTTGGTCAAGAGGAAGAGACCTACAACATCGTGGCAG CCCACGGTTACTTCGGTCGCTTGATCTTCCAATACGCATCGTTCAACAACAGCCGTTC GCTGCACTTCTTCCTGGGTGCATGGCCGGTCGTGGGCATCTGGTTTACCTCCATGGGC ATCAGCACCATGGCGTTCAACCTGAATGGTTTCAACTTCAACCAGTCGGTTTTGGATA GCCAAGGCAAAGTGATCAACACTTGGGCAGATGTGTTGAACCGTGCCAACTTGGGCA TGGAAGTGATGCACGAGCGTAATGCTCACAACTTCCCGCTCGACTTGGCAGCAGGCG AAGCGACCCCGGTCGCTTTtACTGCGCCTTCAATTCACGGTTAA >OE9-L353F (SEQ ID NO: 10) MTSILREQRRDNVWDRFCEWVTSTDNRIYVGWFGVLMIPTLLTATICFIVAFIAAPPVDID GIREPVAGSLMYGNNIISGAVVPSSNAIGLHFYPIWEAASLDEWLYNGGPYQLVVFHFLL GISCYMGRQWELSYRLGMRPWICVAYSAPLSAAFAVFLIYPIGQGSFSDGMPLGISGTFNF MFVFQAEHNILMHPFHMLGVAGVFGGSLFSAMHGSLVTSSLVRETTETESQNYGYKFGQ EEETYNIVAAHGYFGRLIFQYASFNNSRSLHFFLGAWPVVGIWFTSMGISTMAFNLNGFN FNQSVLDSQGKVINTWADVLNRANLGMEVMHERNAHNFPLDLAAGEATPVAFTAPSIH G T354A nucleic acid sequence (SEQ ID NO: 11)Attorney Docket no.10457-590PC0 ATGACCAGCATTCTTCGCGAGCAACGCCGCGATAACGTTTGGGATCGGTTTTGTGAGT GGGTAACCAGCACCGACAACCGCATCTACGTGGGTTGGTTCGGCGTGCTGATGATCC CCACTCTGCTGACCGCCACCATCTGCTTCATCGTTGCGTTCATTGCAGCCCCTCCCGT CGACATCGACGGCATCCGTGAGCCCGTTGCCGGCTCTCTCATGTATGGCAACAACATC ATTTCCGGCGCTGTTGTTCCTTCCAGCAACGCCATCGGCCTGCATTTCTATCCGATTTG GGAAGCCGCTAGCCTCGACGAGTGGCTGTACAACGGTGGTCCTTACCAATTAGTGGT CTTCCACTTCTTGCTGGGTATCAGCTGCTACATGGGTCGTCAATGGGAGCTGTCGTAC CGCCTCGGTATGCGCCCTTGGATCTGTGTTGCATACAGTGCTCCACTCTCGGCTGCTTT TGCAGTGTTTCTGATCTACCCGATCGGCCAAGGTTCGTTCTCGGACGGCATGCCCCTG GGTATCAGCGGCACCTTCAACTTCATGTTCGTGTTCCAAGCAGAGCACAACATTTTGA TGCACCCCTTCCACATGCTGGGTGTGGCTGGTGTGTTCGGTGGTTCGCTGTTCTCGGC AATGCACGGTTCGTTGGTGACCAGCTCGCTGGTGCGTGAGACGACCGAGACCGAGA GCCAAAACTACGGCTACAAATTTGGTCAAGAGGAAGAGACCTACAACATCGTGGCAG CCCACGGTTACTTCGGTCGCTTGATCTTCCAATACGCATCGTTCAACAACAGCCGTTC GCTGCACTTCTTCCTGGGTGCATGGCCGGTCGTGGGCATCTGGTTTACCTCCATGGGC ATCAGCACCATGGCGTTCAACCTGAATGGTTTCAACTTCAACCAGTCGGTTTTGGATA GCCAAGGCAAAGTGATCAACACTTGGGCAGATGTGTTGAACCGTGCCAACTTGGGCA TGGAAGTGATGCACGAGCGTAATGCTCACAACTTCCCGCTCGACTTGGCAGCAGGCG AAGCGACCCCGGTCGCTTTGgCTGCGCCTTCAATTCACGGTTAA T354A amino acid sequence (SEQ ID NO: 12) MTSILREQRRDNVWDRFCEWVTSTDNRIYVGWFGVLMIPTLLTATICFIVAFIAAPPVDID GIREPVAGSLMYGNNIISGAVVPSSNAIGLHFYPIWEAASLDEWLYNGGPYQLVVFHFLL GISCYMGRQWELSYRLGMRPWICVAYSAPLSAAFAVFLIYPIGQGSFSDGMPLGISGTFNF MFVFQAEHNILMHPFHMLGVAGVFGGSLFSAMHGSLVTSSLVRETTETESQNYGYKFGQ EEETYNIVAAHGYFGRLIFQYASFNNSRSLHFFLGAWPVVGIWFTSMGISTMAFNLNGFN FNQSVLDSQGKVINTWADVLNRANLGMEVMHERNAHNFPLDLAAGEATPVALAAPSIH G Ctp nucleic acid sequence (SEQ ID NO: 13)Attorney Docket no.10457-590PC0 ATGATCCAGCCATTTCTGAAGCGATCGCTCCTTTGGCTCACGCTGTTGTTCTGGTTTGC GACAGCGACGCCGGCGTTGGCCCTGACGGAAGAGCAGAAACTGTTCAATGAGGCCT GGCGCATTGTCAACCAGTCCTACGTCGATCCCAGCTTTAACCACAGCAACTGGTTCCA GTTGCGCGAGAAGATTCTGAAAAAGCCGCTGGACAATCGGGATCAGACCTACACCGC GATCGAAGGTTTGCTAGCCAAACTAGATGACCCATTTACGCGGTTGCTACGGCCAGAT CAATATCGCAACCTCCAAGTCTCGACGGCCGGTGAGCTATCGGGCGTCGGTTTGCAG ATTGGCTTTGAGGCCGAAAGTGGCGATGTGGTGGTGATTGCCCCGATCGAAGGTTCG CCGGCAGCCCTGGCGGGTCTGCTGTCGGGCGATCGCATCCTGACGGTGGATGGGGTG GCGATTTCCGGTCGAGATCTGGATGAAGCGGCGGCTCGGATGCGCGGACCTCGCGGG ACGACGGTGGCGCTCCAAGTCCTGCGCGATCAACAAACCTTGGATTTTGAACTAGTT CGCGATCGCATCAGTCTCAATCCCGTGCGATCGCAGCTGGATCGCGATGGCGATCATC CACCGATTGGCTACATTCGCCTCAGCCAATTTAATGCCAATGCCTCGGTAGAAGTGGC TCATGCGATCGCCCAACTCGATCAGCAGGGAGCCGAAGCCTTCGTCCTTGATTTGCGC AATAACTCAGGCGGACTGCTGACCGCTGGCATCGAGATTGCGCGGGAATGGCTGAAC GAAGGCGCGATCGTCTACACCGTCAATCGTCAGGGGGTGCTGGATAGCTTTGCGGCA AATGGCCAAGCGCTGACGGATAAACCCTTGGCACTCCTCGTCAATCGCGGCACTGCT AGCGCCAGCGAAATTCTAGCCGGCGCACTACAAGACAATGAACGCGCCATTTTGGTG GGCGATCGCACCTTTGGCAAAGGTTTGATTCAGTCGCTGTTTGAACTGTCGGATGGG GCGGGTTTGGCGGTCACTGTCGCCAAATACGAAACGCCCAACCACAACGACATCAAT AAGCAGGGCATTCAGCCCGATCTCGCAGTTGAACAGTCTGAACCGCTGTTTGCGGAG GCGATTGCTTCCGCTGCCGATCGCCAATATCAGGCAGCGGTAACGGCGCTGACAGAG CAAGTGAGGCATCGTGCCTAG CtpA amino acid sequence SEQ ID NO: 14 MIQPFLKRSLLWLTLLFWFATATPALALTEEQKLFNEAWRIVNQSYVDPSFNHSNWFQLR EKILKKPLDNRDQTYTAIEGLLAKLDDPFTRLLRPDQYRNLQVSTAGELSGVGLQIGFEA ESGDVVVIAPIEGSPAALAGLLSGDRILTVDGVAISGRDLDEAAARMRGPRGTTVALQVL RDQQTLDFELVRDRISLNPVRSQLDRDGDHPPIGYIRLSQFNANASVEVAHAIAQLDQQG AEAFVLDLRNNSGGLLTAGIEIAREWLNEGAIVYTVNRQGVLDSFAANGQALTDKPLAL LVNRGTASASEILAGALQDNERAILVGDRTFGKGLIQSLFELSDGAGLAVTVAKYETPNH NDINKQGIQPDLAVEQSEPLFAEAIASAADRQYQAAVTALTEQVRHRAAttorney Docket no.10457-590PC0 References 1. 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Plant Cell (2024). 38. Faralli, M. & Lawson, T. Natural genetic variation in photosynthesis: an untapped resource to increase crop yield potential? Plant J. 101, 518-528 (2020). 39. Clapero, V., Arrivault, S. & Stitt, M. Natural variation in metabolism of the Calvin-Benson cycle. Semin. Cell Dev. Biol. 155, 23-36 (2024).Attorney Docket no.10457-590PC0 40. Ungerer, J., Wendt, K. E., Hendry, J. I., Maranas, C. D. & Pakrasi, H. B. Comparative genomics reveals the molecular determinants of rapid growth of the cyanobacterium Synechococcus elongatus UTEX 2973. Proc. Natl. Acad. Sci. U. S. A. (2018). 41. Golden, S. S., Brusslan, J. & Haselkorn, R. Expression of a family of psbA genes encoding a photosystem II polypeptide in the cyanobacterium Anacystis nidulans R2. EMBO J. 5, 2789- 2798 (1986). 42. Ivleva, N. B., Shestakov, S. V. & Pakrasi, H. B. The Carboxyl-Terminal Extension of the Precursor D1 Protein of Photosystem II Is Required for Optimal Photosynthetic Performance of the Cyanobacterium Synechocystis sp. PCC 68031. Plant Physiol. 124, 1403-1412 (2000). 43. Schrader, S. & Johanningmeier, U. The carboxy-terminal extension of the D1-precursor protein is dispensable for a functional photosystem II complex in Chlamydomonas reinhardtii. Plant Mol. Biol. 19, 251-256 (1992). 44. Lers, A., Heifetz, P. B., Boynton, J. E., Gillham, N. W. & Osmond, C. B. The carboxyl- terminal extension of the D1 protein of photosystem II is not required for optimal photosynthetic performance under CO2- and light-saturated growth conditions. J. Biol. Chem. 267, 17494- 17497 (1992). 45. Sheridan, K. J., Duncan, E. J., Eaton-Rye, J. J. & Summerfield, T. C. The diversity and distribution of D1 proteins in cyanobacteria. Photosynth. Res. 145, 111-128 (2020). 46. Zurawski, G., Bohnert, H. J., Whitfeld, P. R. & Bottomley, W. Nucleotide sequence of the gene for the Mr 32,000 thylakoid membrane protein from Spinacia oleracea and Nicotiana debneyi predicts a totally conserved primary translation product of Mr 38,950. Proc. Natl. Acad. Sci. U. S. A. 79, 7699-7703 (1982). 47. Chen, J.-H. et al. Nuclear-encoded synthesis of the D1 subunit of photosystem II increases photosynthetic efficiency and crop yield. Nat. Plants 6, 570-580 (2020). 48. Andrews, S. FastQC: a quality control tool for high throughput sequence data. (2010). 49. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120 (2014). 50. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357-359 (2012). 51. Danecek, P. et al. Twelve years of SAMtools and BCFtools. GigaScience 10 (2021). 52. Seemann, T. Snippy: fast bacterial variant calling from NGS reads. (2018).Attorney Docket no.10457-590PC0 53. Zavřel, T., Sinetova, M. A. & Červený, J. Measurement of Chlorophyll a and Carotenoids Concentration in Cyanobacteria. Bio. Protoc. 5, e1467 (2015).

Claims

Attorney Docket no.10457-590PC0 CLAIMS What is claimed is:

1. A transgenic plant with improved photosynthesis efficiency under salt stress, wherein the plant comprises cells expressing at least one variant of psbA1 gene encoding a variant pre- D1 (pD1) protein, and wherein the variant psbA1 gene is a mutated psbA1 gene having at least one point mutation in a wild type psbA1 gene from cyanobacterium, Synechococcus elongates, and / or optionally from S. elongates PCC 7942 strain.

2. The transgenic plant of claim 1, wherein the wild type psbA1 gene from S. elongatus PCC 7942 comprises a nucleotide sequence of SEQ ID NO:1 encoding a wild type pD1 protein, which comprises an amino acid sequence of SEQ ID NO:

2.

3. The transgenic plant of claim 1 or 2, wherein a variant psbA1 gene having at least one point mutation encodes a variant pD1 protein having at least one missense mutation for amino acid substitution in the carboxyl terminus region near the site to be cleaved by the protease CtpA or near the end of the pD1.

4. The transgenic plant of claim 3, wherein the variant psbA1 gene having a point mutation comprises a nucleotide sequence of SEQ ID NO:3, which encodes a variant pD1 protein having a missense mutation of I358N, and wherein the pD1 I358N comprises an amino acid sequence of SEQ ID NO:

4.

5. The transgenic plant of claim 3, wherein the variant psbA1 gene having a point mutation comprises a nucleotide sequence of SEQ ID NO:5, which encodes a variant pD1 protein having a missense mutation of T354P, and wherein the pD1 T354P comprises an amino acid sequence of SEQ ID NO:

6.

6. The transgenic plant of claim 3, wherein the variant psbA1 gene having a point mutation comprises a nucleotide sequence of SEQ ID NO:7, which encodes a variant pD1 protein having a missense mutation of H359N, and wherein the pD1 H359N comprises an amino acid sequence of SEQ ID NO:

8.

7. The transgenic plant of claim 3, wherein the variant psbA1 gene having a point mutation comprises a nucleotide sequence of SEQ ID NO:9, which encodes a variant pD1 proteinAttorney Docket no.10457-590PC0 having a missense mutation of L353F, and wherein the pD1 L353F comprises an amino acid sequence of SEQ ID NO:

10.

8. The transgenic plant of claim 3, wherein the variant psbA1 gene having a point mutation comprises a nucleotide sequence of SEQ ID NO:11, which encodes a variant pD1 protein having a missense mutation of T354A, and wherein the pD1 T354A comprises an amino acid sequence of SEQ ID NO:

12.

9. The transgenic plant of claim 3, wherein the variant psbA1 gene has 2, 3, or 4 point mutations, which encodes a variant pD1 protein having 2, 3, or 4 missense mutations, and wherein the missense mutations are at least two selected from L353F, I358N, H359N, T354P (or T354A), and optionally L353F+T354A+I358N.

10. The transgenic plant of any of the preceding claims, wherein the plant cells comprising at least one variant of psbA1 gene further comprises ctpA gene comprising a nucleotide sequence of SEQ ID NO:13 encoding CtpA7942 comprising an amino acid sequence of SEQ ID NO:14, and wherein the ctpA gene is from S. elongates PCC 7942 strain.

11. The transgenic plant of any of the preceding claims, wherein the plant is a crop plant or Arabidopsis.

12. A seed that produces the plant of any of claims 1-11.

13. An explant of the plant of any of claims 1-11.

14. A DNA fragment comprising a variant of psbA1 gene having a point mutation, wherein the variant psbA1 gene comprises a nucleotide sequence selected from SEQ ID NOs:3, 5, 7, 9, and 11, which encodes a variant pD1 protein having a missense mutation selected from I358N, T354P, H359N, L353F, and T354A.

15. A DNA fragment comprising a variant of psbA1 gene having at least two point mutations, which encodes a variant pD1 protein having at least two missense mutations selected from I358N, T354P, H359N, L353F, and T354A.

16. The DNA fragment of claim 14 or 15, wherein the DNA fragment comprises an inducible- or constitutively active promoter before the 5’ region of the psbA1 gene.Attorney Docket no.10457-590PC0 17. The DNA fragment of claim 16, wherein the DNA fragment comprises a terminator sequence after the 3’ region of the psbA1 gene.

18. The DNA fragment of claim 14 or 15, wherein optionally the DNA fragment is T-DNA.

19. A plasmid comprising the DNA fragment of claim 14 or 15.

20. Agrobacterium comprising the DNA fragment of claim 14 or 15, or the plasmid of claim 19.

21. The Agrobacterium of claim 20, wherein optionally the bacterium comprises more than one kind of plasmid, and wherein each kind of plasmid comprises a DNA fragment comprising one selected from SEQ ID NOs:3, 5, 7, 9, and 11.

22. The Agrobacterium of claim 20 or 21, wherein the Agrobacterium is Agrobacterium tumefaciens.

23. Cyanobacteria comprising the DNA fragment of claim 14 or 15, or the plasmid of claim 19 for biofuel and alcohol production.

24. The cyanobacteria of claim 23, wherein optionally the bacteria comprise more than one kind of plasmid, and wherein each kind of plasmid comprises a DNA fragment comprising one selected from SEQ ID NOs:3, 5, 7, 9, and 11.

25. The cyanobacteria of claim 23 or 24, wherein the cyanobacteria are the bacteria belonging to the order of Synechococcales, and optionally Synechococcus elongates.

26. A transgenic plant with improved tolerance to salt and light stress, and / or reduced susceptibility to a plant pathogen infection, wherein the plant comprises cells expressing at least one variant psbA1 gene wherein the variant psbA1 gene is a mutated psbA1 gene having at least one point mutation in a wild type psbA1 gene from cyanobacterium, Synechococcus elongates, and optionally from S. elongates PCC 7942 strain, wherein the at least one point mutation is selected from I358N, T354P, H359N, L353F, and / or T354A.

27. The transgenic plant of claim 26, wherein at the at least one variant of psbA1 gene is selected from SEQ ID NOs: 3, 5, 7, 9 and / or 11.Attorney Docket no.10457-590PC0 28. The transgenic plant of claim 26 or 27, wherein the pathogen is fungi or bacteria, and it is optionally Pseudomonas syringae.

29. A method for increasing yield of a plant, the method comprising introducing a genetic modification into cells of the plant that express or causing expression or overexpression of at least one variant of pre-D1 (pD1) protein, wherein the plant comprises cells comprising at least one variant of psbA1 gene encoding the variant pD1 protein, and wherein the variant psbA1 gene is a mutated psbA1 gene having at least one point mutation in a wild type psbA1 gene from cyanobacterium, Synechococcus elongates, and / or optionally from S. elongates PCC 7942 strain into the plant.

30. A method for increasing photosynthesis efficiency in a plant, the method comprising of introducing a genetic modification for causing expression or overexpression of at least one variant of pre-D1 (pD1) protein, wherein the plant comprises cells comprising at least one variant of psbA1 gene encoding the variant pD1 protein, and wherein the variant psbA1 gene is a mutated psbA1 gene having at least one point mutation in a wild type psbA1 gene from cyanobacterium, Synechococcus elongates, and / or optionally from S. elongates PCC 7942 strain into the plant.

31. A method for increasing salt tolerance and / or light tolerance in a plant, the method comprising of introducing a genetic modification for causing expression or overexpression of at least one variant of pre-D1 (pD1) protein, wherein the plant comprises cells comprising at least one variant of psbA1 gene encoding the variant pD1 protein, and wherein the variant psbA1 gene is a mutated psbA1 gene having at least one point mutation in a wild type psbA1 gene from cyanobacterium, Synechococcus elongates, and / or optionally from S. elongates PCC 7942 strain into the plant.

32. A method for reduced susceptibility to a plant pathogen infection in a plant, the method comprising of introducing a genetic modification for causing expression or overexpression of at least one variant of pre-D1 (pD1) protein, wherein the plant comprises cells comprising at least one variant of psbA1 gene encoding the variant pD1 protein, and wherein the variant psbA1 gene is a mutated psbA1 gene having at least one point mutation in a wild type psbA1 gene from cyanobacterium, Synechococcus elongates, and / or optionally from S. elongates PCC 7942 strain into the plant.Attorney Docket no.10457-590PC0 33. The method of any of claims 29-32, wherein the at least one point mutation is selected from I358N, T354P, H359N, L353F, and / or T354A.

34. The method of claim 33, wherein at the at least one variant of psbA1 gene is selected from SEQ ID NOs: 3, 5, 7, 9 and / or 11.

35. A transgenic plant of any of claims 1-11, wherein the at least one point mutation is selected from I358N, T354P, H359N, L353F, and / or T354A.

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