Progress 07/15/18 to 07/14/23
Outputs
Target Audience:The project is designed identify host resistance genes to Gram positive Clavibacter and understand the virulence factors in the bacterial species. The benefits will be realized by corn breeders and producers. Beyond the stake holders, the target audiences are researchers in allied subjects, including important Clavibacter-associated diseases of tomato and potato as well as the Gram positive pathogen Rathybacter toxicus, which is a designated Biological Select Agent. Changes/Problems:The major change for this project is to use Rp1/Rp1 instead of Rp1/rp1 to screen for Rp1 deletion alleles and avirulence bacterial strains. This is due to the partial resistance dominant of individual Rp1 alleles. The change delays the project due to the required analysis of two alleles. We will also conduct additional study for fine-mapping a few QTLs. No major changes are proposed for genetic analysis of virulence factors of Cn. What opportunities for training and professional development has the project provided?Three postdoctoral fellows were supported at Kansas State University, University of Florida, and Iowa State University. The summer 2019 we hired an undergraduate student from the K-State Research and Extension (KSRE) Research Fellowship program, which is a multicultural undergraduate and graduate summer research program for members of ethnic minority groups and other under-representative groups. At KSU, summer intern opportunities have been provided for REEU undergraduate students at KSU in the summer of 2021, 2022, and 2023. The workshop lecture about R programming and RNA-seq analysis have been provided by PI Liu at KSU. Genome sequencing technologies and genome assembly were introduced. One undergraduate student at Iowa State University participated in phenotyping the Cmn mutants on the Rp1 resistant corn line. At UF, the funding has provided opportunities for research associate (1), postdoctoral (2), Master's level graduate (1), visiting scientists (2) and undergraduate (1) research opportunities. The postgraduates have conducted genome comparative studies, gene regulation, and mutational analysis of Cmn. Undergraduate and graduate students have examined Cmn for toxic metabolites and disease comparisons to Cmm How have the results been disseminated to communities of interest?Participant presented workshop "Basic bioinformatics and command-line tools for phytopathologists" at American Society of Phytopathology in 2020, 2021 and 2022 and at the 2023 IS-MPMI conference. The research associate has contributed lecture on Gram+ plant pathogens, including Cmn, to PLP6241 Plant Pathogenic Bacteria, a graduate level class at UF. Cmn genetic mapping work has been presented at the PAG conference and the Genetics of Maize Microbe Interactions seminar. We also presented our CVF1 story and the genetic mapping study in the 2023 IS-MPMI conference. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. to identify the causal gene responsible for GW resistance at the rp1 locus using deletion lines derived from unequal crossovers and confirm the causality by complementation. 1. Phenotype and genotype association Previous years (years 1-4) we have identified 11 disease associated loci through employing multiple genome-wide association strategies. We have validated three loci and identified candidate genes. Validation of the association loci was performed by comparing the phenotypic impacts from different genotypes in near isogenic lines. We have prioritized the genes for further validation: 1) GRMZM2G319357 encoding a low molecular weight protein-tyrosine-phosphatase and responded to bacterial inoculation; 2) GRMZM5G824843 and GRMZM2G330302 that encodes a WD40-repeat containing proteins; 3) GRMZM2G369485 encoding a homolog with rice glutaredoxins GRXS17. This work has been published this year (2023) in G3. 2. Rp1 resistance mapping We have created fine-mapping populations using the Rp1 line as a donor parent and generated BSR-seq (bulked segregant RNA-seq) and GBS (Genotype-by-Sequencing) data to map the resistance loci. The result showed that three genomic loci on chromosomes 2, 4, and 10 were strongly associated with Goss's wilt resistance. During this year, we made an effort to partition each QTL locus. To date, we have developed populations for only segregating at one locus, which will dramatically simply the mapping process. Objective 2. To screen maize Rp1 resistant lines with Cmn (now Cn) mutants to identify compatible bacterial strains, and screen susceptible maize lines to identify non-virulence bacterial strains. Cn mutants have been generated through a treatment with EMS. Individual mutants were used to inoculate maize seedlings, identifying a dozen of non-virulent bacterial strains. These non-virulent bacterial strains have been recovered, confirmed, and sequenced. We have focused on characterization of a virulence gene in Objective 3. At the same time, we found that multiple resistance elements in the Rp1 resistant lines, which complicates the screening of compatible bacterial strains. We are generating maize lines with single-gene resistance for screening bacterial mutants. Objective 3. to sequence compatible and non-virulence bacterial mutant strains, mine candidate avirulence and virulence genes by comparing genomic sequences and validate candidate genes through genetic manipulation. The genomes of three Cn strains were sequenced using PacBio, including one virulent and two non-pathogenic isolates. Genome assembly resulted in three complete genome sequences. The two non-pathogenic isolates were, in fact, isolates of Cn, although not isogenic with each other. Nonetheless, genomic sequence comparisons identified a common virulence factor, referred to as cvf1 (Clavibacter virulence factor 1), which was mutated in the two non-pathogenic isolates in comparison with the pathogenic isolate. A mutation in cvf1 in a wildtype strain resulted in loss of pathogenicity. The altered amino acid residue is assumed to result in either the loss of function, reduced function, or reduced expression of the CVF1 protein, although the specific function of CVF1 is unknown. The wild type cvf1 restored pathogenicity of cvf1 mutants. CVF1 does not have a signal secretion peptide or transmembrane domain, indicating that CVF1 is not directly involved in the interaction with the host. RNA-seq was performed on a wild type (cvf1+) and a mutant (cvf1-), revealing a limited number of genes that are altered in expression between the strains, The comparison indicated that a toxin biosynthesis (herbicidin-related) pathway maybe involved in pathogenicity. Preliminary mutagenesis results indicate that, indeed, pathogenicity requires an intact toxin synthetic pathway. We also note that cvf1 is conserved in all other Clavibacter strains, and homologs of CVF1 are present in other actinobacteria such as Leifsiona, Curtobacteria, and Rathybacter. Some actinobacteria species contain fifteen homologs, suggesting that CVF1 represents a novel regulatory element.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Y Hao, Y Hu, J Jaqueth, J Lin, C He, G Lin, M Zhao, J Ren, TM Tamang, S Park, AE Robertson, FF White, J Fu, B Li, S Liu. (2023). Genetic and transcriptomic dissection of host defense to Gosss bacterial wilt and leaf blight of maize, G3 Genes|Genomes|Genetics, jkad197. doi.org/10.1093/g3journal/jkad197
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
M Zhao, Z Peng, Y Qin, TM Tamang, L Zhang, B Tian, Y Chen, Y Liu, J Zhang, G Lin, H Zheng, C He, K Lv, A Klaus, C Marcon, F Hochholdinger, HN Trick, Y Liu, MJ Cho, S Park, H Wei, J Zheng, FF White, S Liu. (2023). Bacterium-enabled transient gene activation by artificial transcription factors for resolving gene regulation in maize, Plant Cell, 26:koad155. doi.org/10.1093/plcell/koad155
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
N Gyawali, Y Hao, G Lin, J Huang, R Bika, LC Daza, H Zheng, G Cruppe, D Caragea, D Cook, B Valent, S Liu. (2023). Using recurrent neural networks to detect supernumerary chromosomes in fungal strains causing blast diseases, bioRxiv, doi.org/10.1101/2023.09.17.558148
|
Progress 07/15/21 to 07/14/22
Outputs
Target Audience:The project is designed identify hostresistance genes to Gram positive Clavibacter and understand the virulence factors in the bacterial species. The corn growers and industry could benefit from the related discovery. Changes/Problems:The major change for this project is to use Rp1/Rp1 instead of Rp1/rp1 to screen for Rp1 deletion alleles and avirulence bacterial strains. This is due to the partial resistance dominant of individual Rp1 alleles. The change delays the project due to the required analysis of two alleles. We will also conduct additional study for fine-mapping a few QTLs. No major changes are proposed for genetic analysis of virulence factors of Cmn. What opportunities for training and professional development has the project provided?Three postdoctoral fellows were supported at Kansas State University, University of Florida, and Iowa State University. The summer 2019 we hired an undergraduate student from the K-State Research and Extension (KSRE) Research Fellowship program, which is a multicultural undergraduate and graduate summer research program for members of ethnic minority groups and other under-representative groups. At KSU, summer intern opportunities have been provided for REEU undergraduate students at KSU in the summer of 2021 and 2022. The workshop lecture about R programming and RNA-seq analysis have been provided by PI Liu at KSU. Genome sequencing technologies and genome assembly were introduced. One undergraduate student at Iowa State University participated in phenotyping the Cmn mutants on the Rp1 resistant corn line. At UF, the funding has provided opportunities for research associate (1) postdoctoral (2), Master's level graduate (1) and undergraduate (1) research opportunities. The postgraduates have conducted genome comparative studies, gene regulation, and mutational analysis of Cmn. Undergraduate and graduate students have examined Cmn for toxic metabolites and disease comparisons to Cmm. How have the results been disseminated to communities of interest?Participant presented workshop "Basic bioinformatics and command-line tools for phytopathologists" at American Society of Phytopathology in Sept. 2020. The research associate has contributed lecture on Gram+ plant pathogens, including Cmn, to PLP6241 Plant Pathogenic Bacteria, a graduate level class at UF. Cmn genetic mapping work has been presented at the PAG conference. What do you plan to do during the next reporting period to accomplish the goals?This project includes the identification of virulence and avirulence genes from the bacterium, based on annotation of the sequenced Cmn genomes, and identification of the resistance gene from the host. To date, we have found and characterize one virulence gene based on differences in the sequence of naturally occurring closely related virulent and avirulent Cmn strains. We are also generating mutants of homologs of known virulence genes from other pathovars and species of the pathogen. We are preparing a manuscript related to this virulence gene and plan to submit the manuscript. Meanwhile, the manuscript about the genetic mapping is in preparation, which will be submitted soon. Here is the plan: 1. We will focus on identification of Cmn resistance gene in the loci of chromosomes 4 and 10 from the resistance Rp1 donor. We will perform fine mapping of resistance genes from the populations derived from Rp1. 2. We will also perform fine mapping of resistance genes from the populations derived from GA152xA188, in which GA152 is a highly resistance line and A188 is a highly susceptible line. We have performed whole genome sequencing for GA152 and built a reference for A188. The genomic resources would facilitate the fine mapping procedure. 2. We will further elucidate the underlying mechanism of how the virulence gene (cvf1) contributes to the virulence of the bacterium. 3. We will submit two manuscripts: 1) the genetic analysis of the host resistance and 2) the discovery of novel virulence genes and comparative genomics of Clavibacter.
Impacts
What was accomplished under these goals?
2. Rp1 resistance mapping We created fine-mapping populations using the Rp1 line as a donor parent and generated BSR-seq (bulked segregant RNA-seq) and GBS (Genotype-by-Sequencing) data to map the resistance loci. The result showed that two genomic loci, short arms on chromosomes 4 and 10, were strongly associated with Goss's wilt resistance. Efforts have been made to separate these two loci in different sub-populations. Genetic markers of these loci using the KASP technology have been developed for mapping. Objective 2. To screen maize Rp1 resistant lines with Cmn mutants to identify compatible bacterial strains, and screen susceptible maize lines to identify non-virulence bacterial strains. Cmn mutants have been generated through a treatment with EMS. Individual mutants were used to inoculate maize seedlings, identifying a dozen of non-virulent bacterial strains. These non-virulent bacterial strains have been recovered, confirmed, and sequenced. This year we focused on characterization of a non-virulence gene in Objective 3. At the same time, we found that multiple resistance elements in the Rp1 resistant lines, which complicates the screening of compatible bacterial strains. We are generating maize lines with single-gene resistance for screening bacterial mutants. Objective 3. to sequence compatible and non-virulence bacterial mutant strains, mine candidate avirulence and virulence genes by comparing genomic sequences and validate candidate genes through genetic manipulation. The genomes of three avirulent Cmn strains have been sequenced using the PacBio platform. Genome assembly resulted in three complete genome sequences. Sequence analysis identified a virulence factor, referred to as cvf1 (Clavibacter virulence factor 1). A mutation in cvf1 in a wildtype strain resulted in loss of pathogenicity. The altered amino acid residue is assumed to result in either the loss of function, reduced function, or reduced expression of the CVF1 protein, although the specific function of CVF1 is unknown. The wild type cvf1 restored pathogenicity of cvf1 mutants. CVF1 does not have a signal secretion peptide or transmembrane domain, indicating that CVF1 is not directly involved in the interaction with the host. RNA-seq was performed on a wild type (cvf1+) and a mutant (cvf1-), The comparison indicated the herbicidin (a toxin) biosynthesis pathway is involved in the pathogenicity. Note that the cvf1 region is conserved in all other Clavibacter strains, and homologs of CVF1 are present in other actinobacteria such as Leifsiona and Curtobacteria.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
G Lin, H Chen, B Tian, SK Sehgal, L Singh, J Xie, N Rawat, P Juliana, N Singh, S Shrestha, DL Wilson, H Shult, HS Lee, W Adam, VK Tiwari, RP Singh, MJ Guttieri, HN Trick, J Poland, RL Bowden, G Bai, B Gill, S Liu. (2022). Cloning of the broadly effective wheat leaf rust resistance gene Lr42 transferred from Aegilops tauschii. Nat Commun, 13:3044. 10.1038/s41467-022-30784-9
|
Progress 07/15/20 to 07/14/21
Outputs
Target Audience:The project made efforts to identify host resistance genes to Gram+ Clavibacter. The corn growers and industry could benefit from the related discovery. Changes/Problems:The major change for this project is to use Rp1/Rp1 instead of Rp1/rp1 to screen for Rp1 deletion alleles and avirulence bacterial strains. This is due to the partial resistance dominant of individual Rp1 alleles. The change delays the project due to the required analysis of two alleles. We will also conduct additional study for fine-mapping a few QTLs. No major changes are proposed for genetic analysis of virulence factors of Cmn. What opportunities for training and professional development has the project provided?Three postdoctoral fellows were supported at Kansas State University, University of Florida, and Iowa State University. Meanwhile, one graduate student is participating in phenotyping and fine mapping at Kansas State University. The summer 2019 we hired an undergraduate student from the K-State Research and Extension (KSRE) Research Fellowship program, which is a multicultural undergraduate and graduate summer research program for members of ethnic minority groups and other under-representative groups. One undergraduate student at Iowa State University participated in phenotyping the Cmn mutants on the Rp1 resistant corn line. At UF, the funding has provided opportunities for research associate (1) postdoctoral (2), Master's level graduate (1) and undergraduate (1) research opportunities. The postgraduates have conducted genome comparative studies, gene regulation, and mutational analysis of Cmn. Undergraduate and graduate students have examined Cmn for toxic metabolites and disease comparisons to Cmm. How have the results been disseminated to communities of interest?Participant presented workshop "Basic bioinformatics and command-line tools for phytopathologists" at American Society of Phytopathology in Sept. 2020. The research associate has contributed lecture on Gram+ plant pathogens, including Cmn, to PLP6241 Plant Pathogenic Bacteria, a graduate level class at UF. What do you plan to do during the next reporting period to accomplish the goals?This project includes the identification of virulence and avirulence genes from the bacterium, based on annotation of the sequenced Cmn genomes, and identification of the resistance gene from the host. To date, we have found and characterize one virulence gene based on differences in the sequence of naturally occurring closely related virulent and avirulent Cmn strains. We are also generating mutants of homologs of known virulence genes from other pathovars and species of the pathogen. We are preparing a manuscript related to this virulence gene and plan to submit the manuscript before August 30th. Meanwhile, we are preparing the manuscript about the genetic mapping (GWAS and XP-GWAS, QTL). In the next period, we will focus on the identification of Cmn resistance gene in the Rp1 locus and the bacterial genes associated with host resistance. 1. Fine mapping resistance genes from the populations of Rp1/rp1@ and (GA152xA188)@, in which GA152 is a highly resistance line and A188 is a highly susceptible line. We have performed whole genome sequencing for GA152 and built a reference for A188. The genomic resources would facilitate the fine mapping procedure. 2. Elucidating the underlying mechanism of how the virulence gene contributes to the virulence of the bacterium. 3. Submitting two-three manuscripts: the genetic analysis of the host resistance, the discovery of novel virulence genes, and comparative genomics of Clavibacter.
Impacts
What was accomplished under these goals?
Objective 1. to identify the causal gene responsible for GW resistance at the rp1 locus using deletion lines derived from unequal crossovers and confirm the causality by complementation. 1. Rp1 experiments We identified two strong GW resistance alleles or haplotypes (Rp1G and Rp1IG) at the rp1 locus. The crosses of Rp1/Rp1 x rp1/rp1 of both genes were made. The Rp1/rp1 plants carrying heterozygous Rp1G showed resistance but were not as strong as compared to the homozygous Rp1G/Rp1G (and Rp1IG). The proportion of strong resistance in the crosses was ~1/4, while ~1/2 of the crosses showed moderate resistance, indicating both Rp1G and Rp1IG are partial dominant resistance alleles. In addition, QTL analysis using the population of (Rp1 x rp1)@ (@ designates selfing) found that there are probably multiple genes controlling the resistant phenotype, which complicated the analyses of this objective and objective 2 as well. 2. Haplotypes of the rp1 locus Meanwhile, we have characterized the rp1 locus in two high-quality genome assemblies, including our assembly of A188. Both genomes carry >12 rp1 homologous genes, and the gene organizations are fairly different (Figure 1). Nevertheless, the rp1 region is tractable, using current sequencing technologies, which we will use to compare rp1 haplotypes of resistant and susceptible lines upon completion of crosses, and additional deletion and parental lines. ?3. GWAS and XP-GWAS The disease lesion length data of 410 lines were subjected to GWAS to identify GW disease-associated variant loci (DAVs). An alternative genome-wide association strategy, called extreme phenotype GWAS (XP-GWAS) on pools of highly R lines (N=37) and highly S lines (N=44), which were selected from 615 lines, was also employed (Yang et al. 2015). Upon comparing the XP-GWAS results with the standard GWAS analysis, four GW disease-associated loci were co-localized on chromosomes 1, 3, and 8. QTL mapping was performed using three bi-parental recombinant inbred line (RIL) populations, including two nested association mapping (NAM) RIL subpopulations, B73xHP301 and B73xM37W, and the inter-mated B73xMo17 RIL (IBM RIL) population. Combining all mapping results, twelve GW disease-associated loci (gw1a, gw1b, gw1c, gw2a, gw2b, gw2c, gw3a, gw3b, gw4a, gw4b, gw6, and gw8) were identified (Figure 2). The candidate gene analysis identified 11 candidate genes (Table 1). For example, the gene GRMZM2G016439 of gw1a is in the gibberellin signal activation pathway, which is related to the activation of the defense to diseases. Table 1 The candidate genes in the LD block from association loci. Loci Gene ID Chra Start End Gene description q valueb gw1a GRMZM2G016439 1 205,263,817 205,266,907 Histidine-containing phosphotransfer protein 2.91×10-7 gw1b GRMZM2G148281 1 207,946,160 207,950,205 12-oxo-phytodienoic acid reductase 0.003 gw1c GRMZM2G028151 1 281,700,006 281,703,981 AP2-EREBP-transcription factor 184 NA gw2a GRMZM2G125728 2 12,359,037 12,364,897 NA 0.020 gw3a GRMZM5G835677 3 1,406,842 1,410,217 DNAJ heat shock family protein 4.61×10-8 gw3b GRMZM5G824843 3 213,622,361 213,626,336 WD40 repeat-like superfamily protein 0.002 gw5a GRMZM2G161512 5 41,714,515 41,716,637 MYB domain protein 0.002 gw7a GRMZM2G161587 7 43,337,611 43,344,599 NA 0.030 gw7b GRMZM2G363052 7 85,662,465 85,665,508 EREB transponson factor 0.020 gw8a GRMZM2G059497 8 149,851,364 149,863,364 Putative leucine-rich repeat transmembrane protein kinase family protein NA gw10a GRMZM2G143725 10 99,117,687 99,128,736 Zinc finger 7.64×10-7 a, chromosome of the candidate gene b, q values of differential expression analysis in R and S pools 4. RNA-Seq We have performed transcriptomic comparisons between GW resistance and susceptible lines with and without inoculation (or infection) with Clavibacter michiganensis pv. nebraskensis pathogen (Cmn). We have analyzed data and find a list of genes showing bacterial responses (an example in Figure 3) and genes showing differential responses in resistance and susceptible lines. Combination of RNA-Seq with genetic analysis is ongoing. We hope to identify candidate genes to follow up on. 5. Fine mapping of a major QTL We identified a highly resistant line (GA152) and a major QTL and constructed a mapping population, finding three QTLs. We are working on fine mapping of a QTL by phenotyping recombinants that were identified through genotyping. Objective 2. To screen maize Rp1 resistant lines with Cmn mutants to identify compatible bacterial strains, and screen susceptible maize lines to identify non-virulence bacterial strains. Cmn mutants have been generated through a treatment with EMS. Individual mutants were used to inoculate maize seedlings, identifying >10 non-virulent bacterial strains. These non-virulent bacterial strains have been recovered and confirmed. Protocols are also optimized to increase throughput. However, the fact that multiple resistance elements in the Rp1 resistant lines complicates the screening of compatible bacterial strains. We have adjusted our objectives to focus on more on elucidating the resistance mechanisms in the host first. A toal of 672 Cmn mutants were individually screened on seedlings (growth stage V3) of a maize Rp1 resistant line. Six mutants caused lesion lengths at least twice as long as the normal range for the wild-type Cmn strain. These mutants are being confirmed and evaluated on different maize lines. Objective 3. to sequence compatible and non-virulence bacterial mutant strains, mine candidate avirulence and virulence genes by comparing genomic sequences and validate candidate genes through genetic manipulation. The genomes of three avirulent Cmn strains have been sequenced using the Illumina sequencing platform. Sequence analysis revealed a common gene that harbored mutations in two of the three avirulent strains but not in virulent strains, the latter including one newly sequenced virulent Cmn strain as well as genomes of presumed virulent strains in the NCBI database. The candidate virulence gene (gene M) was confirmed to confer bacterial virulence through complementation of the wild type allele from virulent strains in two avirulent strains. The gene is conserved, with a few polymorphisms in some pathovars/species, and encodes a small, hypothetical proteins with no clear conserved domains. Gene M controls expression of at least three operons. Two of the operons contain genes involved in oxidative stress. A third is unknown but may be involved in production of a secondary metabolite. Mutations in the operon have phenotype similar to mutations in gene M. We are currently examining the underlying mechanism. However, based on the preliminary evidence, the results indicate that Cmn has a novel virulence mechanism in comparison to the tomato pathogen Cmm and the potato pathogen Cms. Additional Cmn mutants have been isolated by random transposon mutagenesis and characterization of the mutants and corresponding genes are in progress. None appear to be homologs of known virulence factors of Cmm and Cms.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
G Lin, C He, J Zheng, DH Koo, H Le, H Zheng, D Koo, H Le, H Zheng, TM Tamang, J Lin, Y Liu, M Zhao, Y Hao, F McFarland, B Wang, Y Qin, H Tang, DR McCarty, H Wei, MJ Cho, S Park, H Kaeppler, S Kaeppler, Y Liu, NM Springer, PS Schnable, G Wang, FF White, S Liu. (2021). Chromosome-level genome assembly of a regenerable maize inbred line A188, Genome Biology, 22:175
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
C He, G Lin, H Wei, H Tang, FF White, B Valent, S Liu. (2020). Factorial estimating assembly base errors using k-mer abundance difference (KAD) between short reads and genome assembled sequences, NAR Genomics and Bioinformatics, 2:lqaa075
|
Progress 07/15/19 to 07/14/20
Outputs
Target Audience:Researchers in the fields of Plant-microbe interactions, Plant Genetics and Genomics. Changes/Problems:The major change for this project is to use Rp1/Rp1 instead of Rp1/rp1 to screen for Rp1 deletion alleles and avirulence bacterial strains. This is due to the partial resistance dominant of individual Rp1 alleles. The change delays the project due to the required analysis of two alleles. We will also conduct additional study for fine-mapping a few QTLs. What opportunities for training and professional development has the project provided?Three postdoctoral fellows were supported at Kansas State University, University of Florida, and Iowa State University. Meanwhile, one graduate student is participating in phenotyping and fine mapping at Kansas State University. The summer 2019 we hired an undergraduate student from the K-State Research and Extension (KSRE) Research Fellowship program, which is a multicultural undergraduate and graduate summer research program for members of ethnic minority groups and other under-representative groups. One undergraduate student at Iowa State University participated in phenotyping the Cn mutants on theresistant maize line. How have the results been disseminated to communities of interest?Talks about the progress of this project was presented at the PAG conference meeting in 2019, and two invited lectures were presented at the workshop "Gram-Positive Plant-Associated Bacteria" at the IS-MPMI XVIII Congress in Glasgow, Scotland. What do you plan to do during the next reporting period to accomplish the goals?This project includes the identification of virulence and avirulence genes from the bacterium, based on annotation of the sequenced Cn genomes, and identification of the resistance gene from the host. To date, we have found and characterize one virulence gene based on differences in the sequence of naturally occurring closely related virulent and avirulent Cn strains. We are also generating mutants of homologs of known virulence genes from other pathovars and species of the pathogen. We are preparing a manuscript related to this virulence gene and plan to submit the manuscript before August 30th. Meanwhile, we are preparing the manuscript about the genetic mapping (GWAS and XP-GWAS, QTL). In the next period, we will focus on the identification of Cn resistance gene in the Rp1 locus and the bacterial genes associated with host resistance. 1. Preparation of a bulk of Rp1/Rp1 seeds (~5,000 seeds), which are highly resistant to Cn. The seeds will be ready in November 2020. 2. Screen for moderate resistance that are likely to be Rp1/rp1 (a deletion allele), confirm, and sequence the lines. (Dec 2020 - May 2021) 3. Screen EMS bacteria to find bacterial strains that overcome the resistance. (Dec 2020-March 2021). 4. Associate bacterial mutations with altered host gene expression.
Impacts
What was accomplished under these goals?
1. Objective 1. 1.1. Rp1 experiments We identified two strong GW resistance alleles or haplotypes (RpG and Rp-IG) at the rp1 locus. The crosses of Rp1/Rp1 x rp1/rp1 of both genes were made (Rp1 represent either RpG or Rp-IG). The Rp1/rp1 plants carrying heterozygous Rp1showed resistance but were not as strong asthe homozygous Rp1/Rp1. The proportion of strong resistance in the F2 crosses was ~1/4, while ~1/2 of the crosses showed moderate resistance, indicating both RpG and Rp-IG are partial dominant resistance alleles. 1.2. Haplotypes of the rp1 locus Meanwhile, we have characterized the rp1 locus in two high-quality genome assemblies, including our assembly of A188. Both genomes carry >12 rp1 homologous genes, and the gene organizations are fairly different. Nevertheless, the rp1 region is tractable using current sequencing technologies, which we will use to compare rp1 haplotypes of resistant and susceptible lines upon completion of crosses, and additional deletion lines from the screen (Objective 2). 1.3. GWAS and XP-GWAS The disease lesion length data of 410 lines were subjected to GWAS to identify GW disease-associated variant loci. An alternative genome-wide association strategy, called extreme phenotype GWAS (XP-GWAS) on pools of highly R linesand highly S lines, which were selected from 615 lines, was also employed. Upon comparing the XP-GWAS results with the standard GWAS analysis, four GW disease-associated loci were co-localized on chromosomes 1, 3, and 8. QTL mapping was performed using three bi-parental recombinant inbred line (RIL) populations, including two nested association mapping (NAM) RIL subpopulations, B73xHP301 and B73xM37W, and the inter-mated B73xMo17 RIL (IBM RIL) population. Combining all mapping results, twelve GW disease-associated loci (gw1a, gw1b, gw1c, gw2a, gw2b, gw2c, gw3a, gw3b, gw4a, gw4b, gw6, and gw8) were identified. 1.4. RNA-Seq We have performed transcriptomic comparisons between GW resistance and susceptible lines with and without inoculation (or infection) with Clavibacter nebraskensis pathogen (Cn). Note that the species name has been changed from Cmn to Cn.We have analyzed data and find a list of genes showing bacterial responses and genes showing differential responses in resistance and susceptible lines. Combination of RNA-Seq with genetic analysis is ongoing. We hope to identify candidate genes to follow up on. 1.5. Fine mapping of a major QTL We identified a highly resistant maize line (non-Rp1 line)and a major QTL and constructed a mapping population, finding three QTLs. We are working on fine mapping of a QTL by phenotyping recombinants that were identified through genotyping. 2. Objective 2. Cn mutants have been generated through a treatment with EMS. Individual mutants were used to inoculate maize seedlings, identifying >10 non-virulent bacterial strains. These non-virulent bacterial strains have been recovered and confirmed. Compatible bacterial strains will be screened when the bulk seeds of Rp1/Rp1are ready. Protocols are optimized to increase throughput. We anticipate the screening from early next year. Meanwhile, atoal of 672 Cn mutants were individually screened on seedlings (growth stage V3) of another non-Rp1 maize resistant line. Six mutants caused lesion lengths at least twice as long as the normal range for the wild-type Cn strain. These mutants are being confirmed and evaluated on different maize lines. 3. Objective 3. The genomes of three non-virulent Cn strains have been sequenced using both PacBio and Illumina sequencing platform. Sequence analysis revealed a common gene (geneM) that harbored mutations in two of the three avirulent strains but not in virulent strains, the latter including one newly sequenced virulent Cn strain as well as genomes of presumed virulent strains in the NCBI database. The candidate virulence gene was confirmed to confer bacterial virulence through complementation of the wild type allele from virulent strains in two non-virulent strains. The gene is conserved, with a few polymorphisms in some pathovars/species, and encodes a small, hypothetical proteins with no clear conserved domains. We are currently examining the underlying mechanism.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Peng, Z., Oliveira-Garcia, E., Lin, G., Hu, Y., Dalby, M., Migeon, P., & Liu, S. (2019). Effector gene reshuffling involves dispensable mini-chromosomes in the wheat blast fungus. PLOS Genetics, 15, e1008272.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
He, C., Du, Y., Fu, J., Zeng, E., Park, S., White, F., & Liu, S. (2020). Early drought-responsive genes are variable and relevant to drought tolerance. G3, g3.401199.2020.
|
Progress 07/15/18 to 07/14/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three postdoctoral fellows were supported at Kansas State University, University of Florida, and Iowa State University. Meanwhile, one graduate student is participating in phenotyping and fine mapping at Kansas State University. This summer we hired an undergraduate student from the K-State Research and Extension (KSRE) Research Fellowship program that is a multicultural undergraduate and graduate summer research program for members of ethnic minority groups and other under-representative groups. How have the results been disseminated to communities of interest?A talk about the progress of this project was presented at the PAG conference meeting in 2019. What do you plan to do during the next reporting period to accomplish the goals?This project includes the identification of virulence genes and avirulence genes from the bacterium and identification of the resistance gene from the host. To date, we have found a virulence gene. In the second year we will finish the work related to this virulence gene and publish the study. Meanwhile, we will start to screen for avirulence bacterial gene(s) and host resistance genes. We hope to have both candidate genes in the beginning of the third year of the project. We would like to leave time for validation.
Impacts
What was accomplished under these goals?
Objective 1. to identify the causal gene responsible for GW resistance at therp1locus using deletion lines derived from unequal crossovers, and confirm the causality by complementation. We identified GW resistance alleles (Rp1) at the rp1 locus. The crosses of Rp/Rp1 x rp1/rp1 were made. The progeny Rp1/rp1 have been confirmed to be resistant. This summer, we will produce >5000 seeds of Rp1/rp1. The screening will start late this year. Meanwhile, we identified and confirmed another QTL locus showing a strong effect on GW resistance. The locus was located on the chromosome 1 and fine mapping is currently ongoing. Objective 2. to screen maizeRp1resistant lines with Cmn mutants to identify compatible bacterial strains, and screen susceptible maize lines to identify non-virulence bacterial strains. Bacterial Cmn mutants have been generated through a treatment with EMS. Individual mutants were used to inoculate maize seedlings, identifying >10 non-virulence bacterial strains. These non-virulence bacterial strains have been recovered and confirmed. We have not started the screening for compatible bacterial strains. Protocols are optimized to increase throughput. We anticipate the screening from early next year. Objective 3. to sequence compatible and non-virulence bacterial mutant strains, mine candidate avirulence and virulence genes by comparing genomic sequences, and validate candidate genes through genetic manipulation. The non-virulence bacterial strains were whole-genome sequenced with the Illumina sequencing platform. Sequence analysis found a common gene harboring mutations in all non-virulence bacterial strains but not in virulence strains. This candidate gene was confirmed to confer bacterial virulence through complementation. We are currently attempting to examine the underlying mechanism.
Publications
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