Free and / or nanostructured modified antisense oligonucleotide, compositions, methods and use for the control of citrus cancer

Description

Free and / or nanostructured modified antisense oligonucleotide, compositions, methods and use for the control of citrus canker. Field of invention

[001] The present invention belongs to the technical sector of plant biotechnology, plant protection and genetic control of agricultural diseases. More specifically, it relates to the development and use of antisense oligonucleotides for gene silencing aimed at controlling citrus canker. The invention can also be applied in the sectors of plant defense technologies based on gene silencing. The invention refers to a free or nanostructured antisense oligodeoxynucleotide (ASO) for silencing the Lateral Organ Boundaries 1 (LOB 1) gene, genes regulated by it and other susceptibility genes in citrus plants, aiming at controlling citrus canker. The technology can be applied to various species and varieties of citrus affected by the disease, including oranges, lemons and tangerines, since the underlying genetic mechanism — activation of LOB1 by the bacterium — is conserved among different citrus host species. Fundamentals of the invention

[002] Citrus species are among the most widespread fruit crops in the world and play a significant socio-economic role, especially in countries like Brazil. Brazil holds 50% of the world's orange juice production, exports 98% of what it produces, and collects approximately 190 million dollars in taxes for the country (Neves et al., 2021 https: / / citrusbr.com / biblioteca / publicacoes-citrusbr / o-retrato-da-citricultura / ). Despite the competitiveness of the national citrus sector, orchard productivity is constantly affected by pests and diseases that generate economic and environmental losses (Neves et al. Global orange juice market: a 16-year summary and opportunities for creating value. Trop. plantpathol. 2020; 45, 166-174 https: / / doi.org / 10.1007 / s40858-020-00378-1).

[003] Among the diseases affecting citrus farming, citrus canker stands out as one of the oldest, whose incidence has been progressively increasing, compromising Brazilian productivity (https: / / www.fundecitrus.com.br / levantamentos; Behlau, F. An overview of citrus canker in Brazil. Trop. plant pathol. 2021; 46, 1-12 https: / / doi.org / 10.1007 / s40858-02000377-2). Citrus canker is a disease caused by the bacterium Xanthomonas citri subsp. citri Petition 870250039941, dated 05 / 15 / 2025, page 9 / 46 2 / 13 (X. citri), which injects the effector protein PthA4 into the plant cell, responsible for activating the expression of the LOB1 gene. This gene encodes a transcription factor that induces the expression of genes associated with cell wall loosening, facilitating the entry and colonization of the pathogen. As LOB1 is considered a plant susceptibility gene, silencing it reduces the severity of the disease (Su et al., 2023, Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a / crRNA ribonucleoprotein in the TO generation. Nat Commun. 2023; 14(1): 3957 doi: 10.1038 / s41467-023-39714-9.). Thus, the present invention proposes the application of ASOs to prevent the activation of LOB1, and consequently the genes regulated by it, since LOB1 is a transcription factor, minimizing the damage caused by citrus canker. In the state of São Paulo, the increased incidence of the disease has motivated the adoption of control strategies aimed at reducing the spread of the bacterium X. citri.However, there is no effective control for citrus canker once the disease is established (https: / / www.fundecitrus.com.br / doencas / cancro). Currently, the use of sprays with copper-based products is recommended. However, copper is a heavy metal, and increased application can cause environmental damage, such as contamination of groundwater, as well as phytotoxicity in plants and increased bacterial resistance (Lamichhane et al. Thirteen decades of antimicrobial copper compounds applied in agriculture. A review. Agron. Sustain. Dev. 2018; 38, 28 https: / / doi.org / 10.1007 / s13593-0180503-9). This scenario highlights the urgent need for more sustainable control alternatives to ensure Brazil maintains its leading role in the citrus agribusiness.

[004] An innovative approach under study involves the use of antisense DNA molecules to achieve gene silencing. This strategy utilizes antisense oligonucleotides (ASOs) containing phosphorothioate-type chemical modifications, which can target plant susceptibility genes or pathogen virulence genes. ASOs are short (approximately 20 bp) single-stranded polymers of DNA or RNA, synthesized in vitro, that regulate gene expression by specifically binding to the target RNA sequence, inducing its cleavage by the action of the RNase H enzyme. The phosphorothioate modifications confer high stability in the cellular environment, preventing degradation by plant nucleases (e.g., phosphatases). Thus, this methodology presents important advantages over the use of conventional dsRNAs. Petition 870250039941, dated 05 / 15 / 2025, page 10 / 46 / 13

[005] ASO-based technology has been predominantly explored in the medical field (Geary et al., 2015, Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv Drug Deliv Rev. 2015; 87:46-51. doi: 10.1016 / j.addr.2015.01.008; Chery, 2016, RNA therapeutics: RNAi and antisense mechanisms and clinical applications. Postdoc J. 2016; 4(7):35-50; US20190264200A1), with some studies directed towards the control of agricultural pests or applications in plants (Dinç et al., 2011, Synthetic antisense oligodeoxynucleotides to transiently suppress different nucleus- and chloroplast-encoded proteins of higher plant chloroplasts. Plant Physiol.2011; 157(4):1628-41, Oberemok et al. 2018; A Half-Century History of Applications of Antisense Oligonucleotides in Medicine, Agriculture and Forestry: We Should Continue the Journey. Molecules. 2018. 23(6). pii: E1302. doi: 10.3390 / molecules23061302; WO2024 / 100038; PI 0508559-4).Recently, patent WO2024 / 100038 was filed, targeting genes of plant pathogens. In the present invention, the target genes are from the plant and lack specificity, and therefore may result in resistance to other pathogens. Product regulation is simpler since it is not characterized as an agrochemical (which targets the pathogen). However, the greatest challenge to the success of this tool lies in its efficient absorption by cells and its durability in the environment. Thus, the development of more efficient delivery methods is fundamental to increasing the effectiveness of gene silencing and enabling the creation of new products, such as nanoformulations, aimed at controlling plant diseases.

[006] In this context, the ability of nanoparticles to cross biological membranes has driven the development of nanocarriers, initially for animal cells and, more recently, for plant cells. Nanoparticle-based delivery methods offer advantages such as low cytotoxicity, wide applicability to various plant species, protection of encapsulated molecules against enzymatic degradation, and controlled release in specific organelles or tissues. Thus, the combination of gene silencing strategies with nanotechnology allows the development of nanoformulations with high efficiency and low toxicity to living organisms and the environment, representing a promising approach for pest and disease control in agriculture.

[007] This invention proposes a citrus canker control strategy based on the use of antisense oligodeoxynucleotides (ASOs) active in silencing the Lateral Organ Boundaries 1 (LOB1) gene associated with susceptibility in citrus. LOB1 is a member of the family of Petition 870250039941, dated 05 / 15 / 2025, page 11 / 46 / 13 LOB transcription factors, known to positively regulate genes associated with cell expansion (de Souza-Neto et al., 2023, The Expansin Gene CsLIEXPI Is a Direct Target of CsLOB1 in Citrus. Phytopathology. 2023; 113(7):1266-1277. doi: 10.1094 / PHYTO-11-220424-R.). This gene is considered a susceptibility gene to Xanthomonas citri subsp. citri, since its promoter region has already been reported to contain the binding sequence (EBE) recognized by the bacterial effector PthA4. PthA4 functions as a transcription factor that positively regulates plant genes to facilitate colonization by bacteria (Abe & Benedetti 2016. Additive roles of PthAs in bacterial growth and pathogenicity associated with nucleotide polymorphisms in effector-binding elements of citrus canker susceptibility genes. Mol Plant Pathol. 2016; 17:1223-1236).

[008] Due to the importance of LOB1 in colonization by Xanthomonas citri subsp. Regarding the genes it regulates, such as EXPANSIN, the use of technologies like CRISPR-Cas9 for editing these genes has been explored as an alternative for controlling the disease (Jia et al., 2022, Editing of the LOB1 Promoter via CRISPR / Cas9 Creates Canker-Resistant 'Duncan' Grapefruit. Phytopathology. 2022; 112(2):308-314. doi: 10.1094 / PHYTO-04-21-0144-R.; Jia et al., 2024, Generation of transgene-free canker-resistant Citrus sinensis cv. Hamlin in the T0 generation through Cas12a / CBE co-editing. Front Plant Sci. 2024. doi: 10.3389 / fpls.2024.1385768; de Souza-Neto et al., 2025, CRISPR / Cas9-Mediated Disruption of CsLIEXPI Reveals Expansin as a Key Susceptibility Factor for Citrus Canker Disease. Mol Plant Microbe Interact. 2025 doi: 10.1094 / MPMI-12-24-0151-R).Although CRISPR gene editing technology, applied to the LOB1 gene and genes regulated by it, such as EXPANSIN, represents a potential strategy for developing resistance to citrus canker, the generation of edited plants is a technically complex and lengthy process, especially in perennial species. After editing, a prolonged period is necessary for plant growth, seedling production, field testing, and validation that the permanent silencing of target genes does not compromise vegetative development and productivity. Only after the completion of these steps would large-scale multiplication and gradual availability of seedlings to the production sector be possible, which implies a period of several years until commercial fruit production.Furthermore, although countries like Brazil, the United States, and England recognize plants edited without the insertion of exogenous DNA (DNA-free) as non-transgenic, in the European Union these plants are. Petition 870250039941, dated 05 / 15 / 2025, page 12 / 46 / 13 regulated as genetically modified organisms, restricting the commercial acceptance of fruits and derived products.

[009] The present invention proposes an alternative based on the use of ASOs, which presents technical advantages, allowing the transient silencing of gene expression without the need for permanent genetic alterations. This approach enables the implementation of citrus canker control strategies in a faster, more sustainable way, compatible with different international regulations, meeting the demands of the agricultural and environmental markets. Additionally, the nanostructuring of ASOs promoted strong plant protection against the pathogen, allowing the use of concentrations approximately ten times lower, resulting in a significant reduction in the costs associated with the application of the technology. Brief description of the figures Figure 1. A total of 4 plants were used per treatment, in addition to the controls, nano-free and water, respectively. After 48 h of treatment infiltration, X.citri_GFP was inoculated onto random leaves using a pin-roller to cause micro-injuries on the abaxial surface of the leaves, and the inoculum was applied with the aid of sterile cotton. The leaves were collected at specific times and stored in a -80°C freezer. Phenotypic analysis of the symptoms was performed using photographic records and fluorescence microscopy with GFP light. Subsequently, gene expression analysis of the CsLOBI gene was performed by qRT-PCR. Figure 2. Schematic of cancer symptom quantification. At least four internal replicates are analyzed for cancer lesions by measuring pixels using ImageJ software. Image taken from supplementary material of the article NASCIMENTO et al., (2022). Figure 3 - Analysis of citrus canker symptoms in Citrus sinensis leaves previously treated with ASO3. A. Representative leaves demonstrating the evolution of citrus canker symptoms with ASO3 treatment compared to its negative control (water). Image recording began from the appearance of the first symptoms and was monitored at specific times. B. Quantification of the symptomatic area using ImageJ software. Asterisks represent a significant difference using the t-student test (P<0.05). Petition 870250039941, dated 05 / 15 / 2025, page 13 / 46 / 13 Figure 4 - Analysis of citrus canker symptoms in Citrus sinensis leaves previously treated with ASO3-Nano. A. Representative leaves demonstrating the evolution of citrus canker symptoms with ASO3-Nano treatment compared to its Nano-free control. Image recording began from the appearance of the first symptoms and was monitored at specific times. B. Quantification of the symptomatic area using ImageJ software. Asterisks represent a significant difference using the Student's t-test (* P<0.05; ** P< 0.01). Figure 5 - Analysis of citrus canker symptoms in Citrus sinensis leaves previously treated with ASO3 and ASO3-Nano. A. Representative leaves demonstrating the evolution of citrus canker symptoms with ASO3-Nano treatment compared to ASO3. Image recording began from the appearance of the first symptoms and was monitored at specific times. B. Quantification of the symptomatic area using ImageJ software. Asterisks represent a significant difference using the Student's t-test (* P<0.05; ** P< 0.01). Figure 6 - Quantification of the symptomatic area using ImageJ software. Asterisks represent a significant difference using the t-student test (* P<0.05; ** P< 0.01). Figure 7. Cslobl gene expression evaluated by qRT-PCR at times 1, 2, 3, and 4 after inoculation with X.citri compared to time 0 (before inoculation). A. After treatment with ASO3 compared to the control inoculated with water. B. After treatment with ASO3-Nano compared to the Nano-free control. C. Comparison of ASO3 with ASO3-Nanostructured. The asterisks show statistically significant differences for Cslob1 gene repression using Student's t-test (*P < 0.05; ***P < 0.001). Figure 8 - Evaluation of bacterial growth of leaves treated with ASO3, ASO3-Nano, Nano-free and water. Differences between means were calculated using the Student's t-test (P<0.05). Detailed description of the invention Petition 870250039941, dated 05 / 15 / 2025, page 14 / 46 / 13

[010] The present invention relates to the development and use of modified, free or nanostructured antisense oligonucleotides (ASOs) for gene silencing of Citrus susceptibility genes for the control of citrus canker. More specifically, the invention relates to the development and use of chemically modified, free or nanostructured antisense oligonucleotides (ASOs) for silencing the LOB 1 susceptibility gene, reducing the damage caused by citrus canker. The active molecule is a single-stranded DNA nucleic acid that can be used in formulations, such as a foliar spray. The antisense sequence (ASO) is complementary to a part of a plant susceptibility gene, which causes the downregulation of the target mRNA through the activation of the RNase H enzyme, leading to the silencing of said gene.

[011] The modified antisense oligonucleotides of the present invention are characterized by being constructed from the LOB 1 gene (SEQ ID NO 1)

[012] The invention relates to sequences that have at least 90%, more specifically 95%, more specifically 98%, more specifically 99% sequence similarity to any of the sequences described, but not limited to: SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ Petition 870250039941, dated 05 / 15 / 2025, p. 15 / 46 / 13 ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121, SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO 131, SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141, SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151, SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161.

[013] The present invention is characterized by the fact that the antisense oligonucleotides are synthesized with first-generation chemical modifications of the phosphorothioate type with the substitution of oxygen atoms by sulfur, but not limited to: other first, second or third generation modifications. The modifications were carried out on the two phosphodiester bonds at the 3' and 5' ends, but not limited to: additional modifications or modifications at other positions of the ASO.

[014] The present invention is also related to compositions for controlling citrus canker using free or nanostructured modified antisense oligonucleotides, as described in this patent application, and an agronomically acceptable carrier. The selection of the sequence for the assays and nanostructuring was based on the GC content in the range of (40-60%) and lower binding energy [Overall (ΔΘ)], and therefore SEQ ID 15 was used in the in planta assays and subsequently for nanostructuring. More specifically, the composition is related to the following agronomically acceptable carriers: chitosan crosslinked by ionic gelation with sodium tripolyphosphate followed by the addition of polyethylene glycol.

[015] The carrier nanoparticles were obtained from the following steps:: a. Synthesize the antisense oligonucleotide as described in the invention; b. Solubilize the natural biopolymer in an acidified aqueous reaction medium. Petition 870250039941, dated 05 / 15 / 2025, page 16 / 46 / 13 c. Add the oligonucleotides selected in “a” to the solution obtained in “b” to a concentration of 0.1 to 10 μM. d. Add a sodium tripolyphosphate solution to the solution obtained in step “c” to achieve ionic gelation. e. Maintain the suspension obtained in “d” under magnetic stirring. f. Add to the solution obtained in “e” polyethylene glycol

[016] The nanoparticle obtained is characterized by the fact that the natural biopolymer is selected from, but not limited to: chitosan, alginate, carrageenan, gellan gum, gelatin, casein, agarose, hydroxypropylmethylcellulose and several others. Also, but not limited to these, synthetic polymers such as polyethylene glycol (PEG), poly(acrylic acid) (PAA), poly(vinyl alcohol) (PVA), polymers containing carboxylates, among others, may be used. More specifically, the present invention relates to the biopolymer chitosan and the copolymer PEG. As a form of nanostructuring, the methods that can be employed include, but are not limited to, gelation, precipitation, complex coacervation, solvent emulsification / evaporation, electrospinning, electrospraying, supercritical techniques, microfluidics, self-assembly and complex formation with molecules and other nanomaterials.As crosslinking agents, monovalent (Na+ and K+), divalent (Ca2+, Ba2+, Mg2+) or trivalent (Fe3+ and Al3+) cations can be used, depending on the case, for anionic polymers such as alginate, pectin, and carrageenan; monovalent anions (chloride and acetate), divalent anions (sulfate and citrate), polyphosphates or polyanions (sodium tripolyphosphate, sodium hexametaphosphate, and tricalcium phosphate) for cationic polymers such as chitosan; other ionic crosslinking agents such as borate ions, copper ions, and zinc ions. More specifically, the present invention relates to the biopolymer chitosan gelled with sodium tripolyphosphate.

[017] The present application also describes the use of modified antisense oligonucleotides, as described in the patent application, characterized by the fact that it is for the control of citrus canker. Petition 870250039941, dated 05 / 15 / 2025, page 17 / 46 / 13

[018] The oligonucleotides, nanoparticles and compositions of the invention can be applied to citrus plants in various forms, including liquid, powder, or as suspensions, and can be applied by spraying, injection, root absorption or other appropriate application methods, depending on the specific need.

[018] Citrus plants that may benefit from the present invention include, but are not limited to: Citrus sinensis, Citrus reticulata, Citrus aurantium, Citrus bergamia, Citrus limon, Citrus grandis, Citrus fortunella mitis, Citrus australasica, Citrus medica, Citrus paradisi, Citrus macrophylla, including different cultivars and hybrids of the genus Citrus.

[019] The present invention is illustrated by the following examples, which are intended to demonstrate its applications, without, however, limiting its scope in any way. In the following examples, references will be made to the figures described above. Examples of embodiments of the invention 1. Comparative tests of free and nanostructured ASO3

[020] Nanoparticles containing ASO3 (SEQ ID NO15), named ASO3-Nano, were characterized by dynamic light scattering and Zeta potential. Average hydrodynamic diameter (Z-average) of 420.2 ± 63.1 nm, polydispersity index (PdI) of 0.743 ± 0.068 and Zeta potential of 31.30 ± 0.80 mV were obtained. In addition, a range of diameter variation of the detected nanostructures was obtained based on the hydrodynamic diameter distribution curves, ranging from 28.2 to 955.4 nm. As a control, for the empty (white) nanoparticles without ASO, the average hydrodynamic diameter (Z-average) obtained was 420.2 ± 63.1 nm, a polydispersity index (PdI) of 0.743 ± 0.068, and a Zeta potential of 31.30 ± 0.80 mV. Furthermore, a range of diameter variation was obtained for the detected nanostructures based on the hydrodynamic diameter distribution curves, ranging from 24.4 nm to 396.1 nm.A total of 4 plants were used for each of the treatments (ASO3-Nano, ASO3, nanostructures without ASO3, and water). Two distinct experiments were conducted. In both experiments, a concentration of 1 μM (of both ASO3s) was used. This concentration is 10 times lower than the concentration used in the ASO3 experiments where inhibition of citrus canker symptoms occurred (presented in previous reports). The 1 μM concentration was recommended by CENARGEN partners, as they had already verified the molecule's higher absorption efficiency. Petition 870250039941, dated 05 / 15 / 2025, page 18 / 46 / 13 A concentration of 10⁸ CFU / mL of the bacterium X. citri_GFP was inoculated onto C. sinensis leaves 48 hours after infiltration of the treatments. For this, the leaves were perforated on their abaxial surface using a pin-roller containing 540 microneedles. Then, the bacterial solution was applied to the micro-injuries using a moistened sterile cotton swab (Figure 1). 2. Assay of LOB1 gene expression to validate the potential of free and nanostructured ASO3 to silence the target gene.

[021] For gene expression assessment, leaves were collected at specific times: T0 (before infiltration and inoculation), T1 (2 days after symptom appearance), T2 (4 days after symptom appearance), T3 (6 days after symptom appearance), and T4 (8 days after symptom appearance). Total RNA was extracted using the PureLink™ RNA Mini Kit (Invitrogen, Waltham, MA, USA) and cDNA was synthesized using the GoScript™ Reverse Transcriptase kit (Promega, Madison, WI, USA). qPCR was performed using SYBR Green, and relative expression was analyzed using the actin gene as a normalizer gene, using the ΔΔCT calculation (KJ LIVAK AND TD SCHMITTGEN. Analysis of relative gene expression data using real-time quantitative PCR and the 2-δδct method. Methods, 25(4):402-408, 2001). 3. Analysis of citrus canker symptomatology in the presence and absence of treatments with free and nanostructured ASO3.

[022] Symptoms were assessed by photographic records and fluorescence microscopy using a GFP filter. Symptom quantification was performed using ImageJ software according to the methodology described in NASCIMENTO et al., (2022) (NASCIMENTO, CA “Overexpression of CsSAMT in Citrus sinensis Induces Defense Response and Increases Resistance to Xanthomonas citri subsp. citri” Frontiers in Plant Science, vol. 13, 2022.) (Figure 2). For this, 4 areas (quadrants) of each leaf were recorded, quantified, and analyzed. The software performs a quantification within a specified area of ​​the quadrant, determining how much of this area corresponds to symptoms, returning a value in pixels per square micrometer (pixels^m2). CFU counting was performed by serial dilution using leaves collected as soon as the first symptoms were detected.

[023] In order to conduct a time course of symptom development, photographic records were made of symptomatic leaves at each of the previously described time points (Figure 1). Through quantitative data on lesion areas of four leaves per Petition 870250039941, dated 05 / 15 / 2025, page 19 / 46 / 13 treatment, a significant reduction in symptoms was observed in leaves treated with ASO3 when compared to the water control (Figure 4). This result was somewhat expected, since we had already observed this reduction in previous trials. However, it is worth noting that in the results presented in Figure 4, a concentration of 1 uM was used, while in the results presented in previous reports, a concentration of 10 uM was used. An even greater significant reduction was observed in relation to leaves treated with ASO3-Nano when compared to the Nano-free control (Figure 5), as well as when compared to ASO3 (Figure 6). These results prove the greater efficiency of ASO3-Nano in interfering with the X. citri - host molecular interaction, consequently reducing the symptoms of the disease. 4. Comparative analysis of the efficiency of free ASO3 vs. ASO3-Nano

[024] Comparing the treatments together, a highly significant reduction in symptoms was observed with the ASO3-Nano treatment compared to the Nano-free negative control (P = 0.0016) and compared to ASO3 (P = 0.0010) (Figure 7). Considering that the concentration of ASO3-Nano is ten times lower than the indicated concentration of 10 uM (Dinç et al., Synthetic antisense oligodeoxynucleotides to transiently suppress different nucleus- and chloroplast-encoded proteins of higher plant chloroplasts. Plant Physiol.157(4):1628-41.2011; Sandoval-Mojica et al., Antibacterial FANA oligonucleotides as a novel approach for managing the Huanglongbing pathosystem. Scientific Reports. https: / / doi.org / 10.1038 / s41598-021-82425-8), and also that used in previous experiments with ASO3, we can conclude that the nano-structuring of this molecule is more effective for gene silencing of CsLOB1 and consequent reduction of citrus canker symptoms.To test this hypothesis, LOB1 expression was evaluated at different times after infiltration of ASO3, ASO3-Nano, and their respective controls.

[025] qRT-PCR analysis was performed comparing the expression of the Cslob1 gene in uninfected leaves collected immediately before inoculation (T0) with different times after treatments with ASO3, ASO3-Nano and their respective controls (Figure 1). The expression of CsLOB1 in the ASO3 treatments was significantly lower than the control treatment (Figure 8A), suggesting that even at a concentration of 1 µM, ASO3 inhibited the expression of this gene. The repression of Cslob1 in the ASO3-Nano treatments was highly significant compared to the control treatment (Figure 8B) and the ASO3 treatment. Petition 870250039941, dated 05 / 15 / 2025, page 20 / 46 / 13 (Figure 8C). These results reinforce the hypothesis that the reduction in citrus canker symptoms observed mainly after treatment with ASO3-Nano is a consequence of the greater efficiency of this molecule in silencing the CsLOB1 gene. 5. Evaluation of the bacterial population

[026] Despite observing a significant reduction in citrus canker symptoms, no significant difference was observed in the bacterial population in the treatments with ASO3 or ASO3-Nano compared to the controls (Figure 9). These data are in agreement with those recently observed by Su et al (2023) (SU, H., WANG, Y., XU, J., OMAR, AA, GROSSER, JW, CALOVIC, M., et al. 2023. Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a / crRNA ribonucleoprotein in the T0 generation. Nat Commun. 14:3957), where they used CRISPR-Cas12a technology to edit the CsLOB1 gene (the same one used in our experiments) of C. sinensis. Similar to our results, the authors found that the edited plants showed a significant reduction in citrus canker, although the number of bacteria was not significantly altered.It has already been demonstrated that overexpression of the CsLOB1 gene in citrus alters plant physiology and induces pustules similar to citrus canker, even without the presence of the bacteria (ZOU, X. et al. CsLOB1 regulates susceptibility to citrus canker through promoting cell proliferation in citrus. The Plant Journal, v. 106, n. 4, p. 1039-1057, 2021). The authors conclude that the symptoms of citrus canker result from the overexpression of CsLOB1 induced by the X. citri effector, and that without expression of this gene, the bacterium survives in the plant, but without causing severe symptoms (SU, H., WANG, Y., XU, J., OMAR, AA, GROSSER, J. W, CALOVIC, M., et al. 2023. Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a / crRNA ribonucleoprotein in the T0 generation).

Claims

1. MODIFIED ANTISENSE OLIGONUCLEOTIDE (ASOs), characterized by comprising at least 90%, more specifically 95%, more specifically 98%, more specifically 99% sequence similarity with any of the selected sequences from the group, but not limited to: SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53,SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121, SEQ ID NO 122,SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID Petition 870250039941, dated 05 / 15 / 2025, page. 22 / 46 2 / 3 NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO 131, SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141, SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151, SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161., 2. MODIFIED ANTISENSE OLIGONUCLEOTIDE (ASOs), according to claim 1, characterized in that the modification is of the phosphorothioate type.

3. MODIFIED ANTISENSE OLIGONUCLEOTIDE (ASOs), according to claim 1, characterized in that the sequence is preferably SEQ ID NO 15.

4. NANOPARTICLE characterized by comprising a natural biopolymer and / or a synthetic polymer and an oligonucleotide described in any one of claims 1 to 3.

5. NANOPARTICLE according to claim 4 characterized in that the natural biopolymer is selected from the group: chitosan, alginate, carrageenan, gellan gum, gelatin, casein, agarose, hydroxypropylmethylcellulose.

6. NANOPARTICLE according to claim 5 characterized in that the natural biopolymer is chitosan.

7. NANOPARTICLE according to claim 4 characterized in that the synthetic polymer is selected from the group: polyethylene glycol (PEG), poly(acrylic acid) (PAA), poly(vinyl alcohol) (PVA), carboxylate-containing polymers 8. NANOPARTICLE according to claim 7 characterized in that the synthetic polymer is polyethylene glycol. Petition 870250039941, dated 05 / 15 / 2025, page 23 / 46 3 / 3 9. COMPOSITION FOR CONTROLLING CITRUS CANCER characterized by comprising an oligonucleotide as described in any one of claims 1 to 3 or a nanoparticle described in any one of claims 4 to 8.

10. METHOD FOR OBTAINING A NANOPARTICLE, characterized by comprising the following steps: a. Synthesizing the antisense oligonucleotide according to any one of claims 1 to 3; b. Solubilizing the natural biopolymer in an acidified aqueous reaction medium; c. Adding the oligonucleotide selected in a to the solution obtained in “b” to a concentration of 0.1 to 10 μM; d. Adding a polyanion solution to the solution obtained in c to achieve ionic gelation; e. Maintaining the suspension obtained in d under magnetic stirring; f. Adding synthetic polymer to the solution obtained in e.

11. METHOD FOR OBTAINING A NANOPARTICLE, according to claim 10, characterized in that the natural biopolymer is a cationic biopolymer such as chitosan.

12. METHOD FOR OBTAINING A NANOPARTICLE, according to claim 10, characterized in that the synthetic polymer is polyethylene glycol 13. USE OF MODIFIED ANTISENSE OLIGONUCLEOTIDE (ASOs), as defined in any one of claims 1 to 3, characterized by being for the control of citrus canker.

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