TaMP gene and application thereof in improving wheat resistance to rust

Abstract

This invention discloses TaMP Genes and their application in improving wheat rust resistance belong to the field of plant biotechnology and breeding. TaMP The nucleotide sequence of the gene is shown in SEQ ID NO.1. This invention utilizes Agrobacterium-mediated wheat embryo genetic transformation technology to prepare... TaMP Transgenic wheat plants with gene overexpression and knockout. It was verified that overexpression... TaMP Genes that reduce wheat's resistance to stripe rust; knockout TaMP The gene enhances wheat's resistance to stripe rust. Therefore, this invention verifies the efficacy of wheat myristylated modified protein. TaMP Genes play a crucial role in wheat's resistance to stripe rust. As mentioned above, TaMP This gene plays a negative regulatory role in the defense response of wheat against rust. It can be used to improve the disease resistance traits of wheat and other crops, and to breed new transgenic plant varieties resistant to disease.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology and breeding, and particularly to... TaMP Genes and their application in improving wheat's resistance to rust. Background Technology

[0002] wheat( Triticum aestivum L. is one of the most widely distributed, cultivated, and commercialized food crops globally. It is produced by stripe rust fungi (… Puccinia striiformis f. sp. Tritici , Pst ) and leaf rust fungi ( Puccinia triticina , Pt Wheat rust, caused by pathogens, severely impacts wheat yield and quality. Planting resistant varieties is currently the most environmentally friendly and effective disease control measure; however, varietal resistance can be overcome by mutations in the pathogen's virulence, leading to re-outbreaks. Therefore, continuously discovering new wheat rust-resistant genes and creating durable resistant varieties is the fundamental way to control wheat rust.

[0003] In plant-pathogen interactions, post-translational modifications of proteins play a crucial regulatory role. N-terminal myristoylation is an irreversible covalent lipid modification of proteins that precisely regulates the subcellular localization and biological function of proteins on the cell membrane through myristyltransferases. Previous studies have shown that myristoylation is involved in plant growth and development, signal transduction, and responses to various abiotic stresses. In the field of plant immunity, some myristoylated proteins have been shown to participate in the transduction of defense signals as immune components.

[0004] However, research on whether myristylated proteins can regulate wheat rust resistance remains very limited. In particular, the existence of a specific class of myristylated proteins that act as "susceptibility factors" during pathogen infection, suppressing the immune response by hijacking normal host physiological pathways, is still a blank area. Therefore, identifying novel susceptibility-associated myristylated proteins (TaMP) and verifying their value in disease resistance improvement will not only help elucidate the molecular nature of wheat susceptibility but also provide important technical support for improving broad-spectrum resistance to stripe rust and leaf rust in wheat through gene editing technology. Summary of the Invention

[0005] The purpose of this invention is to provide TaMP This invention verifies the role of genes and their application in improving wheat rust resistance to address the problems existing in the prior art. TaMP This gene plays a negative regulatory role in the defense response of wheat against rust. It can be used to improve the disease resistance traits of wheat and other crops, and to breed new transgenic plant varieties resistant to disease.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides TaMP Genes, the ones mentioned TaMP The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] The present invention also provides knockout of the aforementioned TaMP The application of gene-based preparations in any of the following: (1) Improve wheat's resistance to rust; (2) To prepare products that enhance wheat's resistance to rust; (3) Breeding rust-resistant wheat; (4) Prepare products for cultivating rust-resistant wheat.

[0008] The present invention also provides a reduction in the aforementioned TaMP The use of formulations that express proteins encoded by genes in any of the following: (1) Improve wheat's resistance to rust; (2) To prepare products that enhance wheat's resistance to rust; (3) Breeding rust-resistant wheat; (4) Prepare products for cultivating rust-resistant wheat.

[0009] The present invention also provides a method for knocking out the aforementioned TaMP The application of gene recombinant vectors in any of the following: (1) Improve wheat's resistance to rust; (2) To prepare products that enhance wheat's resistance to rust; (3) Breeding rust-resistant wheat; (4) Prepare products for cultivating rust-resistant wheat.

[0010] The present invention also provides the use of recombinant microorganisms containing the recombinant vector in any of the following: (1) Improve wheat's resistance to rust; (2) To prepare products that enhance wheat's resistance to rust; (3) Breeding rust-resistant wheat; (4) Prepare products for cultivating rust-resistant wheat.

[0011] Optionally, the rust resistance includes resistance to stripe rust and leaf rust.

[0012] The present invention also provides a method for improving the resistance of wheat to rust, comprising knocking out the aforementioned [unclear] in wheat. TaMP Steps to enhance wheat's resistance to rust through genetic engineering.

[0013] Optionally, the rust resistance includes resistance to stripe rust and leaf rust.

[0014] The present invention also provides a method for breeding rust-resistant wheat, comprising knocking out the aforementioned [unclear] in wheat. TaMP Genes, reducing the aforementioned TaMP The expression level of rust-resistant wheat was determined by the steps involved.

[0015] Optionally, the rust resistance includes resistance to stripe rust and leaf rust.

[0016] The present invention discloses the following technical effects: This invention validates the wheat myristylation modified protein. TaMP Gene expression profiles under stripe rust infection, from which gene expression can be derived. TaMP Expression induced by wheat stripe rust. Furthermore, the gene... TaMP The proteins it encodes are associated with wheat's immunity against stripe rust.

[0017] Through Agrobacterium-mediated genetic transformation of wheat embryos, obtain TaMP Transgenic wheat plants with gene overexpression and knockout. It was verified that overexpression... TaMP Genes that reduce wheat's resistance to stripe rust; knockout TaMP The gene enhances wheat's resistance to stripe rust. Therefore, this invention verifies the efficacy of wheat myristylated modified protein. TaMP Genes play a crucial role in wheat's resistance to stripe rust.

[0018] As mentioned above, TaMP This gene plays a negative regulatory role in the defense response of wheat against rust. It can be used to improve the disease resistance traits of wheat and other crops, and to breed new transgenic plant varieties resistant to disease. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 for TaMP Gene expression patterns in wheat stripe rust (CYR34, CYR23) infection; Figure 2 for TaMP Gene overexpression plants and knockout plants were successfully created; A: In the overexpression plants TaMP Gene expression levels; B: In knockout plants TaMP Gene editing status; Figure 3 for TaMP Gene overexpression reduces wheat resistance to stripe rust; A: Disease incidence in leaves of overexpressing plants after infection with stripe rust fungus; B: Statistical graph of leaf response phenotype after stripe rust fungus infection; C: Stripe rust fungus biomass level in leaves of overexpressing plants; D: Quantitative analysis of ROS accumulation in leaves of overexpressing plants. Figure 4 for TaMP Gene knockout plants enhance wheat resistance to stripe rust; A: Leaf phenotype of gene knockout plants after stripe rust infection; B: Statistical graph of disease severity in leaves after stripe rust infection; C: Biomass level of stripe rust in leaves of gene knockout plants; D: Quantitative analysis of ROS accumulation in leaves of gene knockout plants; E: Relative expression level of disease resistance genes in gene knockout plants; F: Statistical graph of hyphal length and infection area in gene knockout plants after stripe rust infection; Figure 5 for TaMP Gene knockout plants enhance wheat resistance to leaf rust; A: Phenotypic image of gene knockout plants after inoculation with leaf rust fungus strain PKTS; B: Quantitative detection of relative fungal biomass of gene knockout plants after inoculation with leaf rust fungus strain PKTS. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Stripe rust fungi CYR23 and CYR34 were provided by the Wheat Research Institute of Sichuan Agricultural University, and leaf rust fungi PKTS were provided by the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences.

[0027] The nucleotide sequence of the wheat cardamomylated protein TaMP gene is shown in SEQ ID NO.1, and the amino acid sequence of the wheat cardamomylated protein TaMP is shown in SEQ ID NO.2.

[0028] SEQ ID NO.1: ATGGGATCCGTCGCGGCTAGCTCTGCTCACCTCAGGGGCCGCTGTGGCATGCCCAACCGTGTCCTTGGCCGGCGGCAAGATGGGAGCCATCACGGCTAGTGGTGCCCGTCTCCAGCGCACCCAAGGCCATGCCAAGGTTCTGTCCAGCCCGCTCCGT GGAGATGGTGCCATGGTGGAACTGCTGCACCGTCAGCCGATGGCCGAGATGCCGCGCAGTTGCACTAGAAGGTGCGAGCTTGATGCAGACTCAACAGGAAAAGGAGCAAGAACATACCTACAGACGATGAAGTGCCAGGAGCTCCGTCCATGCTGA.

[0029] SEQ ID NO.2: MGSVAASSAHLRGAVACPTVSLAGGKMGAITASGARLQRTQGHAKVLSSPLRGDGAMVELLHRQPMAEMPRSCTRRCELDADSTGKGARTYLQTMKCQELRPC.

[0030] Example 1: Stripe rust fungus infection TaMP Gene expression pattern analysis The wheat variety Fielder was cultured in an incubator at 16℃ with a light / dark cycle of 16 h / 8 h. When the wheat reached the two-leaf-one-heart stage, it was inoculated with stripe rust fungi CYR23 and CYR34: stripe rust fungi soaked in an appropriate amount of sterile water were applied evenly to the wheat leaves with a brush, and the leaves were cultured in the dark at 10℃ for 24 h, followed by normal culture.

[0031] After inoculation with stripe rust, wheat leaves were collected at 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, 72 h, and 120 h. Two leaf segments, approximately 2 cm in length, were taken from each sample and placed in a 2 mL centrifuge tube containing a steel ball (5 mm in diameter). All consumables and reagents used in the experiment, including the steel ball, centrifuge tubes, and pipette tips, were de-RNase-free. Total RNA was extracted from the samples according to the method provided by the TransZol Up Plus RNA Kit. Reverse transcription was performed on the extracted RNA samples, following the method provided by the reverse transcription kit (Aikerui Biotechnology). Using the cDNA as a template, quantitative real-time PCR was performed using the Aikerui Biotechnology premixed qPCR mix. The upstream primer TaMP-DL-F sequence was TGGTGCCATGGTGGAACTGCTGCA (SEQ ID NO.3), and the downstream primer TaMP-DL-R sequence was TCGTCTGTAGGTATGTTCTTGCTC (SEQ ID NO.4). Amplification parameters were set according to the kit instructions. The wheat samples were then subjected to qRT-PCR. TaMP Gene expression profiles were analyzed in response to stripe rust fungus.

[0032] The results are as follows Figure 1 As shown, TaMP The gene response to stripe rust fungus was strong, with significant upregulation in the early stages of infection (especially 12-24 hpi). In the early stages of CYR34 infection, its expression was approximately 1-fold upregulated compared to the control, indicating... TaMP It may play a regulatory role in the early stages of the wheat-rust fungus interaction.

[0033] Example 2: Creation of TaMP transgenic plants (1) TaMP Construction of gene overexpression recombinant plasmids Will TaMP The nucleotide sequence of the gene (SEQ ID NO.1) was amplified, and SEQ ID NO.1 was cloned into the pCAMBIA3301 vector using homologous recombination technology to construct a recombinant plasmid.

[0034] The steps for constructing the overexpression recombinant plasmid were as follows: Using Premier 5 software, specific primers were designed and amplified against the sequence shown in SEQ ID NO.1. The primers used were: TaMP-3301-F: TGCAGCCCGGGGATCCATGGGATCCGTCGCGGCTAGCTCTGC (SEQ ID NO.5), and TaMP-3301-R: ATTCACACGTGCAGATCCTCTTCTGAGATGAGTTTTTGTTCGCATGGACGGAGCTC (SEQ ID NO.6). The amplified products were ligated into the linearized cloning vector pCAMBIA3301, and the experiment was performed using the Universal Bio 2×GenRec Recombinant Kit. E. coli competent cells DH5α were thawed on ice, and the above reaction solution was added to the competent cells for transformation. The bacterial culture was evenly spread on LB agar plates containing appropriate antibiotics and incubated overnight at 37°C. Single colonies were picked for PCR positive detection. The positive single colonies were shaken and sequenced, and the sequencing results were compared with the reference sequence. Select positive clones that have been verified by sequencing and inoculate them into 5-15 mL of LB medium supplemented with the corresponding antibiotics. Then, place the medium in a constant temperature shaker at 37°C and 220 r / min for 16 h of shaking culture. Collect the bacterial culture and extract plasmids according to the instructions of the Qingke Biopharmaceutical Plasmid Extraction Kit to obtain the overexpression recombinant plasmid.

[0035] (2) TaMP Gene editing and recombinant plasmid construction According to wheat TaMP The requirements for gene genome sequence and CRISPR / Cas9 technology target design were considered. Targets containing NGG sites were screened from the WheatCrispr website (https: / / crispr.bioinfo.nrc.ca / WheatCrispr / ). TaMP The gene fragment was then submitted to the Ensembl Plant platform (http: / / plants.ensembl.org / Multi / Tools / Blast) for Blast homology testing to knock out TaMP-7A in the wheat genome. Two gRNAs were designed as gene editing targets within the conserved CDS region of TaMP-7A: sgDNA1 sequence GGCATGCCCAACCGTGTCCT (SEQ ID NO.7) and sgDNA2 sequence GCCCGTCTCCAGCGCACCCA (SEQ ID NO.8).

[0036] The steps for constructing gene-editing recombinant plasmids are as follows: Following the steps described above for constructing overexpression recombinant plasmids, complementary oligonucleotide pairs of sgDNA1 and sgDNA2 with BsaI sticky ends were synthesized and annealed to form double-stranded fragments. The annealed sgDNA1 product was first ligated to the pHUE411 vector using BsaI and T4 DNA ligase, and then transformed into *E. coli*. After screening and sequencing verification, the sgDNA1-pHUE411 intermediate vector was obtained. Using this intermediate vector as a backbone, the annealed sgDNA2 product was ligated into the vector using the same method, ultimately obtaining a recombinant plasmid containing two gRNA expression cassettes tandemly on the same vector, namely the TaMP-7A dual-target gene-editing vector.

[0037] (3) Agrobacterium-mediated genetic transformation of wheat callus The recipient material, the wheat variety Fielder, was planted with a photoperiod of 24℃ for 16 hours and a darkperiod of 20℃ for 8 hours. Seeds were collected 14-16 days after flowering, and immature embryos were isolated. The constructed overexpression recombinant plasmid and gene-editing recombinant plasmid were transformed into Agrobacterium EAH105. Positive single spots were selected and inoculated into MGL liquid medium, and cultured overnight in the dark at 28℃. The collected bacterial suspension was resuspended in the infection solution and added to the immature embryos of the recipient material for infection. After two days of co-culture, the hypocotyl of the immature embryos was removed. After recovery culture, selection culture, differentiation culture, and rooting culture, transgenic positive plants were obtained. The progeny of the transgenic positive plants were then tested and sequenced.

[0038] The results are as follows Figure 2 As shown, TaMP Overexpression in plants TaMP The gene expression level was significantly higher than that of the control group, while TaMP Nucleotide deletions were present in the Cas9 target region of all knockout plants, leading to frameshift mutations. The results indicate... TaMP Overexpression plants (TAMP-OE) and TaMP The TAMP-KO knockout method has been successfully created.

[0039] Example 3 TaMP Identification of stripe rust resistance in plants with overexpressed genes To explore TaMP Whether it participates in the host wheat's immune response during stripe rust infection, and its cultivation. TaMP Overexpression lines were harvested, seeds were collected and sown after maturity, and stable overexpression T1 generation lines were cultured. Seeds from the T1 generation lines were harvested and sown again to obtain T2 generation transgenic material. Disease resistance was assessed on the T2 generation transgenic material containing TaMP, which underwent molecular identification. TaMPOverexpressing plants and the control variety Fielder were cultured in a greenhouse until they reached the stage of two leaves and one bud. Following Example 1, the second leaf was inoculated with stripe rust fungus CYR23, and samples were taken at different time points for observation and quantitative detection.

[0040] On day 14 post-inoculation, leaf disease incidence was observed. 120 h post-inoculation, the relative fungal biomass of sporulating leaves was analyzed by RT-qPCR. TaEF and PstEF were used to represent the biomass of wheat and stripe rust, respectively. The primers used were: TaEF-F: TGGTGTCATCAAGCCTGGTATGGT (SEQ ID NO. 9), TaEF-R: ACTCATGGTGCATCTCAACGGACT (SEQ ID NO. 10); PstEF1-F: TTCGCCGTCCGTGATATGAGACAA (SEQ ID NO. 11), PstEF1-R: ATGCGTATCATGGTGGTGGAGTGA (SEQ ID NO. 12).

[0041] At 24 h and 48 h post-inoculation, the reactive oxygen species (ROS) content in plant tissues was determined using luminol chemiluminescence immunoassay. Samples were obtained using a punch to ensure uniform sample size. Samples were placed in 96-well plates, with 200 μL of sterile water added to each well, and incubated overnight in the dark to eliminate background ROS signals caused by physical damage. A working reaction solution containing 200 μM luminol and 50 μg / mL horseradish peroxidase was prepared. After removing the sterile water from the plate, the above reaction solution was added to each well. If ROS generation is required, 100 μM chitin can be added. Immediately after sample addition, the plate was placed in a microplate reader, and the signal acquisition frequency was set to once per minute for continuous detection for 30 minutes.

[0042] The results are as follows Figure 3 As shown, after inoculation with stripe rust fungus CYR23, TaMP The density of uredinia produced on the leaves of gene-overexpressing plants was significantly higher than that of wild-type controls, with a significant increase in disease severity and a significant increase in stripe rust fungal biomass; histological observation showed... TaMP The accumulation of ROS was significantly reduced in plants that overexpressed the gene. The results indicate that overexpression... TaMP Genes reduce wheat's resistance to stripe rust.

[0043] Example 4 TaMP Identification of stripe rust resistance in gene knockout plants Referring to Example 3, for TaMP After knockout plants were inoculated with stripe rust fungus CYR34, disease phenotypes were observed, disease severity was statistically analyzed, and fungal biomass and reactive oxygen species accumulation were quantitatively measured. RT-qPCR was used to detect the disease at 24 h and 48 h. TaMPKnock out disease resistance genes in plants TaPR 1. TaPR 2. Relative expression level. The detection method was the same as in Example 1. The primers used were: TaPR1-F: GAGAATGCAGACGCCCAAGC (SEQ ID NO.13), TaPR1-R: CTGGAGCTTGCAGTCGTTGATC (SEQ ID NO.14); TaPR2-F: AGGATGTTGCTTCCATGTTTGCCG (SEQ ID NO.15), TaPR2-R: AAGTAGAATGCGCATGCCGTTGATG (SEQ ID NO.16).

[0044] In addition, 24 h and 48 h after inoculation, infected wheat leaves were cut into segments of about 2 cm and placed in a decolorizing solution, then incubated at 37°C until the leaf segments were completely decolorized. The leaves were then rinsed three times with 50 mM Tris-HCl (pH=7.5) buffer, 10 min each time. Finally, 20 μg / mL wheat germ agglutinin (WGA) fluorescent staining solution was added, and the leaves were stained in the dark for 24 h. The length and area of ​​the hyphae were observed and counted under a fluorescence microscope.

[0045] The results are as follows Figure 4 As shown, after inoculation with stripe rust fungus CYR34, TaMP Knocking out spore masses on plant leaves significantly reduced compared to Fielder, resulting in a marked decrease in disease severity. Five days after inoculation, TaMP The fungal biomass in the knockout strains was significantly reduced by approximately 55%. At 24 h and 48 h post-inoculation, TaMP The accumulation of reactive oxygen species was significantly increased in the knockout lines. Analysis of defense-related gene expression showed that... TaMP Knockout strains TaPR1 and TaPR2 Transcriptional levels increased by 50-80%. Histological observation and statistical analysis further indicated that, compared to wild-type Fielder, TaMP The mycelial length and infection area of ​​the knockout plants were significantly reduced. The results indicated that knockout... TaMP Genes enhanced wheat's resistance to stripe rust.

[0046] Example 5 TaMP Identification of leaf rust resistance in gene knockout plants Referring to Example 3, for TaMP Knockout plants were inoculated with the leaf rust fungus strain PKTS, and plant phenotypes were observed 8 days later and detected by RT-qPCR. TaMP Relative fungal biomass in gene knockout plants.

[0047] The results are as follows Figure 5As shown, after inoculation with the leaf rust strain PKTS, TaMP Knockout plants showed a reduction in the number of urediniospores on their leaves, more pronounced yellowing around lesions, and a 55%-70% reduction in leaf rust biomass compared to the Fielder control. The results indicated that knockout... TaMP Genes enhanced wheat's resistance to leaf rust.

[0048] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A kind TaMP Genes, characterized by, The TaMP The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Knockout of claim 1 TaMP The application of gene-based preparations in any of the following: (1) Improve wheat's resistance to rust; (2) To prepare products that enhance wheat's resistance to rust; (3) Breeding rust-resistant wheat; (4) Prepare products for cultivating rust-resistant wheat.

3. Reduce the claim 1 as described in claim 1 TaMP The use of formulations that express proteins encoded by genes in any of the following: (1) Improve wheat's resistance to rust; (2) To prepare products that enhance wheat's resistance to rust; (3) Breeding rust-resistant wheat; (4) Prepare products for cultivating rust-resistant wheat.

4. For knocking out the device described in claim 1 TaMP The application of gene recombinant vectors in any of the following: (1) Improve wheat's resistance to rust; (2) To prepare products that enhance wheat's resistance to rust; (3) Breeding rust-resistant wheat; (4) Prepare products for cultivating rust-resistant wheat.

5. The use of recombinant microorganisms containing the recombinant vector of claim 4 in any of the following: (1) Improve wheat's resistance to rust; (2) To prepare products that enhance wheat's resistance to rust; (3) Breeding rust-resistant wheat; (4) Prepare products for cultivating rust-resistant wheat.

6. The application as described in any one of claims 2-5, characterized in that, The rust resistance includes resistance to stripe rust and leaf rust.

7. A method for improving wheat's resistance to rust, characterized in that, Includes knocking out the wheat as described in claim 1 TaMP Steps to enhance wheat's resistance to rust through genetic engineering.

8. The method as described in claim 7, characterized in that, The rust resistance includes resistance to stripe rust and leaf rust.

9. A method for breeding rust-resistant wheat, characterized in that, Includes knocking out the wheat as described in claim 1 TaMP Genes, reducing the aforementioned TaMP The expression level of rust-resistant wheat was determined by the steps involved.

10. The method as described in claim 9, characterized in that, The rust resistance includes resistance to stripe rust and leaf rust.

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