Pub Date : 2024-08-10DOI: 10.1101/2024.08.09.607401
Karl Effertz, Jonathan K Richards, Shaun James Clare, Madeline del Castillo, Roshan Sharma Poudel, Mengyuan Li, Jianwei Zhang, Matthew James Moscou, Timothy Friesen, Robert Saxon Brueggeman
The foliar disease net form net blotch (NFNB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. teres (Ptt), causes significant yield and quality losses of barley worldwide. Dominant resistance conferred by the Resistance to Pyrenophora teres 5 (Rpt5) gene from barley line CI5791 is the broadest and most effective resistance reported in this pathosystem. The Rpt5 locus was identified in multiple independent genetic studies utilizing diverse host populations and Ptt isolates, and harbors both dominant Rpt5 resistance and isolate-specific susceptibility genes/alleles that are dominant in the absence of Rpt5, designated susceptibility to Pyrenophora teres 1 (Spt1). Ptt virulence and avirulence effectors from diverse pathogen isolates genetically interact with the Rpt5/Spt1 locus, suggesting a complex locus with a function targeted by the evolution of a diversity of pathogen effectors. High-resolution mapping utilizing 1,920 recombinant gametes from a CI5791 x Tifang biparental population, identified 12 candidate genes in an ~4.6 Mb delimited region in the cv Morex V3 genome assembly, but is 1.1 − 2.2 Mb in the pangenome assemblies, containing 5−12 genes. Analysis revealed a strong correlation between the CI5791 allele of a receptor-like protein (RLP), provisionally designated Rpt5 candidate gene 1, (Rcg1), and broad Rpt5−mediated resistance. Two independent transformants of the CI5791 Rcg1 allele in the susceptible cv Golden Promise background showed significantly increased resistance when challenged with Rpt5 avirulent Ptt isolates 6A, 15A, and 0−1 compared to the Golden Promise wildtype. Thus, Rpt5, encodes an RLP and is the first net blotch resistance gene cloned in barley.
{"title":"Rpt5 encodes a receptor-like protein that provides broad and effective net form net blotch (Pyrenophora teres f. teres) resistance in barley","authors":"Karl Effertz, Jonathan K Richards, Shaun James Clare, Madeline del Castillo, Roshan Sharma Poudel, Mengyuan Li, Jianwei Zhang, Matthew James Moscou, Timothy Friesen, Robert Saxon Brueggeman","doi":"10.1101/2024.08.09.607401","DOIUrl":"https://doi.org/10.1101/2024.08.09.607401","url":null,"abstract":"The foliar disease net form net blotch (NFNB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. teres (Ptt), causes significant yield and quality losses of barley worldwide. Dominant resistance conferred by the Resistance to Pyrenophora teres 5 (Rpt5) gene from barley line CI5791 is the broadest and most effective resistance reported in this pathosystem. The Rpt5 locus was identified in multiple independent genetic studies utilizing diverse host populations and Ptt isolates, and harbors both dominant Rpt5 resistance and isolate-specific susceptibility genes/alleles that are dominant in the absence of Rpt5, designated susceptibility to Pyrenophora teres 1 (Spt1). Ptt virulence and avirulence effectors from diverse pathogen isolates genetically interact with the Rpt5/Spt1 locus, suggesting a complex locus with a function targeted by the evolution of a diversity of pathogen effectors. High-resolution mapping utilizing 1,920 recombinant gametes from a CI5791 x Tifang biparental population, identified 12 candidate genes in an ~4.6 Mb delimited region in the cv Morex V3 genome assembly, but is 1.1 − 2.2 Mb in the pangenome assemblies, containing 5−12 genes. Analysis revealed a strong correlation between the CI5791 allele of a receptor-like protein (RLP), provisionally designated Rpt5 candidate gene 1, (Rcg1), and broad Rpt5−mediated resistance. Two independent transformants of the CI5791 Rcg1 allele in the susceptible cv Golden Promise background showed significantly increased resistance when challenged with Rpt5 avirulent Ptt isolates 6A, 15A, and 0−1 compared to the Golden Promise wildtype. Thus, Rpt5, encodes an RLP and is the first net blotch resistance gene cloned in barley.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.07.607017
Arti Rai, Magne Nordang Skårn, Torstein Tengs, Abdelhameed Elameen, Mathias Rudolf Amundsen, Oskar Schnedler Bjorå, Lisa Karine Haugland, Igor A Yakovlev, May Bente Brurberg, Tage Thorstensen
Secondary metabolites produced by the phenylpropanoid pathway, which is regulated by transcription factors of the MYB family, play crucial roles in this early phase of fruit development. The MYB46 transcription factor is a key regulator of secondary cell wall structure and lignin and flavonoid biosynthesis in many plants, but little is known about its activity in flowers and berries in F. vesca. For functional analysis of FvMYB46, we designed a CRISPR-Cas9 construct with an endogenous F. vesca specific U6-promoter for efficient and specific expression of two gRNAs targeting the first exon of FvMYB46. This generated mutants with an in frame 81-bp deletion of the first conserved MYB-domain or an out of frame 82-bp deletion potentially knocking out the gene function. In both types of mutant plants, pollen germination and the frequency of flowers developing to mature berries was significantly reduced compared to wild type. Transcriptomic analysis of flowers demonstrated that FvMYB46 is positively regulating the expression of genes involved in pollen germination, homeostasis of reactive oxygen species (ROS) and the phenylpropanoid pathway, including secondary cell wall biosynthesis and flavonoid biosynthesis, while has a negative impact on carbohydrate metabolism. In FvMYB46-mutant flowers, the flavonols and flavan-3-olscontent, especially epicatechin, quercetin-glucoside and kaempferol-3-coumaroylhexoside were reduced, and we observed a local reduction of lignin content in anthers. Together these results suggest that MYB46 control fertility and efficient fruit set by regulating cell wall structure, flavonoid biosynthesis, carbohydrate metabolism and ROS- signaling in flowers and early fruit development in F. vesca.
{"title":"CRISPR-Cas9 mediated deletions of FvMYB46 reduces fruit set and biosynthesis of flavonoids in Fragaria vesca","authors":"Arti Rai, Magne Nordang Skårn, Torstein Tengs, Abdelhameed Elameen, Mathias Rudolf Amundsen, Oskar Schnedler Bjorå, Lisa Karine Haugland, Igor A Yakovlev, May Bente Brurberg, Tage Thorstensen","doi":"10.1101/2024.08.07.607017","DOIUrl":"https://doi.org/10.1101/2024.08.07.607017","url":null,"abstract":"Secondary metabolites produced by the phenylpropanoid pathway, which is regulated by transcription factors of the MYB family, play crucial roles in this early phase of fruit development. The MYB46 transcription factor is a key regulator of secondary cell wall structure and lignin and flavonoid biosynthesis in many plants, but little is known about its activity in flowers and berries in <em> F. vesca.</em> For functional analysis of FvMYB46, we designed a CRISPR-Cas9 construct with an endogenous <em>F. vesca</em> specific U6-promoter for efficient and specific expression of two gRNAs targeting the first exon of <em>FvMYB46</em>. This generated mutants with an in frame 81-bp deletion of the first conserved MYB-domain or an out of frame 82-bp deletion potentially knocking out the gene function. In both types of mutant plants, pollen germination and the frequency of flowers developing to mature berries was significantly reduced compared to wild type. Transcriptomic analysis of flowers demonstrated that FvMYB46 is positively regulating the expression of genes involved in pollen germination, homeostasis of reactive oxygen species (ROS) and the phenylpropanoid pathway, including secondary cell wall biosynthesis and flavonoid biosynthesis, while has a negative impact on carbohydrate metabolism. In <em>FvMYB46</em>-mutant flowers, the flavonols and flavan-3-olscontent, especially epicatechin, quercetin-glucoside and kaempferol-3-coumaroylhexoside were reduced, and we observed a local reduction of lignin content in anthers. Together these results suggest that MYB46 control fertility and efficient fruit set by regulating cell wall structure, flavonoid biosynthesis, carbohydrate metabolism and ROS- signaling in flowers and early fruit development in <em>F. vesca.</em>","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.08.607113
Prashant Mishra, Shakespear S, Sara Catherine Carpenter, Hamsa Sekar, Vigi S, Anith K N, Prasanta Kumar Dash, Adam J. Bogdanove, Rhitu Rai
To infect rice, Xanthomonas oryzae pv. oryzae (Xoo) deploys transcription activator-like effectors (TALEs) that specifically bind and upregulate host 'susceptibility' (S) genes. 34-amino acid (aa) repeats in TALEs interact one-to-one with DNA bases. Variation at positions 12 and 13 in each repeat, the repeat-variable diresidue (RVD), determine specificity. Some repeat variants shorter or longer than 34 aa can disengage to accommodate a single base deletion in the target sequence. OsSWEET11, 13, and 14 are key S genes, targeted by different TALEs from diverse Xoo strains. xa13 is a SWEET11 allele lacking the TALE binding site and thus conferring resistance. xa13 is overcome by TALEs that activate SWEET13 or SWEET14. We report here that an xa13-compatible Xoo strain, IX-221, from India, harbours an ortholog of the SWEET14-targeting TALE PthXo3 and two orthologs of the SWEET13-cognate PthXo2, each with one or two 36-aa repeats capable of disengaging. One of the PthXo2 orthologs, PthXo2BIX221, has a repeat region identical to the previously characterized PthXo2BPXO61, except for a two amino acid difference near the end of the 19th repeat. Like PthXo2BPXO61, PthXo2BIX221 upregulates SWEET13 in japonica rice and no SWEET in indica rice, but unlike PthXo2B PXO61 it nonetheless renders indica rice susceptible, pointing to an alternative S gene. Further, a designer TALE (dTALE) constructed using a standard, consensus sequence for each repeat and RVDs identical to those of PthXo2BIX221 failed to render indica rice susceptible. Alignment of the PthXo2BIX221 repeats shows a departure from the consensus in each of two repeats carrying the RVD 'NN': the sequence 'MAIAN' in place of 'VAIAS' beginning at position 7. Together, the PthXo2BIX221 results thus suggest that non-RVD sequence variation affects TALE targeting profiles. More broadly, the presence of the three aberrant repeat-harbouring TALEs in IX-221 suggests that widespread deployment of xa13 in India resulted in strains super-equipped to overcome it, capable of activating multiple SWEET genes and alleles as well as an apparent alternate S gene.
为了感染水稻，黄单胞菌（Xanthomonas oryzae pv. oryzae，Xoo）部署了转录激活剂样效应物（TALEs），它们能特异性地结合并上调宿主的 "易感性"（S）基因。TALEs 中的 34 个氨基酸重复序列与 DNA 碱基进行一对一的相互作用。每个重复序列中第 12 和 13 位的变异，即重复序列变异二侧（RVD），决定了特异性。一些短于或长于 34 aa 的重复变体可以脱离，以适应目标序列中的单碱基缺失。OsSWEET11、13 和 14 是关键的 S 基因，是不同 Xoo 菌株的不同 TALE 的靶标。xa13 是 SWEET11 等位基因，缺乏 TALE 结合位点，因此具有抗性。我们在此报告，来自印度的 xa13 兼容 Xoo 菌株 IX-221 含有 SWEET14 靶向 TALE PthXo3 的一个直向同源物和 SWEET13 识别 PthXo2 的两个直向同源物，每个直向同源物都有一个或两个能够脱离的 36-aa 重复序列。其中一个 PthXo2 的直向同源物 PthXo2BIX221 的重复区域与之前表征的 PthXo2BPXO61 相同，只是在第 19 个重复的末端附近有两个氨基酸的差异。与 PthXo2BPXO61 一样，PthXo2BIX221 在粳稻中上调 SWEET13，而在籼稻中不上调 SWEET，但与 PthXo2B PXO61 不同的是，它仍然使籼稻易感，这表明存在另一种 S 基因。此外，利用每个重复的标准共识序列和与 PthXo2BIX221 相同的 RVDs 构建的设计 TALE（dTALE）也不能使籼稻易感。对 PthXo2BIX221 重复序列的比对结果显示，携带 RVD "NN "的两个重复序列中的每一个都偏离了共识：从第 7 位开始，序列 "MAIAN "代替了 "VAIAS"。因此，PthXo2BIX221 的结果表明，非 RVD 序列变异会影响 TALE 的靶向特征。更广义地说，IX-221 中存在的三个异常重复邻接 TALE 表明，xa13 在印度的广泛应用导致菌株具备了克服 xa13 的超级能力，能够激活多个 SWEET 基因和等位基因以及一个明显的替代 S 基因。
{"title":"A PthXo2B ortholog in Xanthomonas oryzae pv oryzae strain IX-221 acts as a major virulence factor on indica rice without activating a Clade III SWEET gene.","authors":"Prashant Mishra, Shakespear S, Sara Catherine Carpenter, Hamsa Sekar, Vigi S, Anith K N, Prasanta Kumar Dash, Adam J. Bogdanove, Rhitu Rai","doi":"10.1101/2024.08.08.607113","DOIUrl":"https://doi.org/10.1101/2024.08.08.607113","url":null,"abstract":"To infect rice, Xanthomonas oryzae pv. oryzae (Xoo) deploys transcription activator-like effectors (TALEs) that specifically bind and upregulate host 'susceptibility' (S) genes. 34-amino acid (aa) repeats in TALEs interact one-to-one with DNA bases. Variation at positions 12 and 13 in each repeat, the repeat-variable diresidue (RVD), determine specificity. Some repeat variants shorter or longer than 34 aa can disengage to accommodate a single base deletion in the target sequence. OsSWEET11, 13, and 14 are key S genes, targeted by different TALEs from diverse Xoo strains. xa13 is a SWEET11 allele lacking the TALE binding site and thus conferring resistance. xa13 is overcome by TALEs that activate SWEET13 or SWEET14. We report here that an xa13-compatible Xoo strain, IX-221, from India, harbours an ortholog of the SWEET14-targeting TALE PthXo3 and two orthologs of the SWEET13-cognate PthXo2, each with one or two 36-aa repeats capable of disengaging. One of the PthXo2 orthologs, PthXo2B<sub>IX221</sub>, has a repeat region identical to the previously characterized PthXo2B<sub>PXO61</sub>, except for a two amino acid difference near the end of the 19th repeat. Like PthXo2B<sub>PXO61</sub>, PthXo2B<sub>IX221</sub> upregulates SWEET13 in japonica rice and no SWEET in indica rice, but unlike PthXo2B <sub> PXO61</sub> it nonetheless renders indica rice susceptible, pointing to an alternative S gene. Further, a designer TALE (dTALE) constructed using a standard, consensus sequence for each repeat and RVDs identical to those of PthXo2B<sub>IX221</sub> failed to render indica rice susceptible. Alignment of the PthXo2B<sub>IX221</sub> repeats shows a departure from the consensus in each of two repeats carrying the RVD 'NN': the sequence 'MAIAN' in place of 'VAIAS' beginning at position 7. Together, the PthXo2B<sub>IX221</sub> results thus suggest that non-RVD sequence variation affects TALE targeting profiles. More broadly, the presence of the three aberrant repeat-harbouring TALEs in IX-221 suggests that widespread deployment of xa13 in India resulted in strains super-equipped to overcome it, capable of activating multiple SWEET genes and alleles as well as an apparent alternate S gene.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941478","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.07.606736
Naoyoshi Kumakura, Takayuki Motoyama, Keisuke Miyazawa, Toshihiko Nogawa, Katsuma Yonehara, Kaori Sakai, Nobuaki Ishihama, Kaisei Matsumori, Pamela Gan, Hiroyuki Koshino, Takeshi Fukuma, Richard J. O'Connell, Ken Shirasu
Many plant pathogenic fungi penetrate host surfaces mechanically, using turgor pressure generated by appressoria, specialized infection cells. These appressoria develop semipermeable cell walls and accumulate osmolytes internally to create turgor by osmosis. While melanin is known to be important for turgor generation, the mechanism for wall semipermeability has remained unclear. Here we identify PKS2 and PBG13, by reverse genetics, as crucial for forming the semipermeable barrier in anthracnose and rice blast fungi. These genes encode enzymes that synthesize 3,5-dihydroxyhexanoic acid polymers essential for the cell wall properties. Deleting these enzymes impairs cell wall porosity, abolishing turgor and pathogenicity without affecting melanization. Our findings uncover a novel mechanism of turgor generation, linking enzyme function to pathogen penetration and disease potential, presenting new targets for disease control.
{"title":"Dihydroxyhexanoic acid biosynthesis controls turgor in pathogenic fungi","authors":"Naoyoshi Kumakura, Takayuki Motoyama, Keisuke Miyazawa, Toshihiko Nogawa, Katsuma Yonehara, Kaori Sakai, Nobuaki Ishihama, Kaisei Matsumori, Pamela Gan, Hiroyuki Koshino, Takeshi Fukuma, Richard J. O'Connell, Ken Shirasu","doi":"10.1101/2024.08.07.606736","DOIUrl":"https://doi.org/10.1101/2024.08.07.606736","url":null,"abstract":"Many plant pathogenic fungi penetrate host surfaces mechanically, using turgor pressure generated by appressoria, specialized infection cells. These appressoria develop semipermeable cell walls and accumulate osmolytes internally to create turgor by osmosis. While melanin is known to be important for turgor generation, the mechanism for wall semipermeability has remained unclear. Here we identify <em>PKS2 and PBG13</em>, by reverse genetics, as crucial for forming the semipermeable barrier in anthracnose and rice blast fungi. These genes encode enzymes that synthesize 3,5-dihydroxyhexanoic acid polymers essential for the cell wall properties. Deleting these enzymes impairs cell wall porosity, abolishing turgor and pathogenicity without affecting melanization. Our findings uncover a novel mechanism of turgor generation, linking enzyme function to pathogen penetration and disease potential, presenting new targets for disease control.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941479","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.08.603566
Youngwoo Lee, Heena Rani, Eileen L Mallery, Daniel B Szymanski
Cotton fibers are aerial trichoblasts that employ a highly polarized diffuse growth mechanism to emerge from the developing ovule epidermis. After executing a complicated morphogenetic program, the cells reach lengths over 2 cm and serve as the foundation of a multi-billion-dollar textile industry. Important traits such as fiber diameter, length, and strength are defined by the growth patterns and cell wall properties of individual cells. At present, the ability to engineer fiber traits is limited by our lack of understanding regarding the primary controls governing the rate, duration, and patterns of cell growth. To gain insights into the compartmentalized functions of proteins in cotton fiber cells, we developed a label-free liquid chromatography mass spectrometry method for systems level analyses of fiber proteome. Purified fibers from a single locule were used to fractionate the fiber proteome into apoplast (APOT), membrane-associated (p200), and crude cytosolic (s200) fractions. Subsequently, proteins were identified, and their localizations and potential functions were analyzed using combinations of size exclusion chromatography, statistical and bioinformatic analyses. This method had good coverage of the p200 and apoplast fractions, the latter of which was dominated by proteins associated with particulate membrane-enclosed compartments. The apoplastic proteome was diverse, the proteins were not degraded, and some displayed distinct multimerization states compared to their cytosolic pool. This quantitative proteomic pipeline can be used to improve coverage and functional analyses of the cotton fiber proteome as a function of developmental time or differing genotypes.
{"title":"A cell fractionation and quantitative proteomics pipeline to enable functional analyses of cotton fiber development","authors":"Youngwoo Lee, Heena Rani, Eileen L Mallery, Daniel B Szymanski","doi":"10.1101/2024.08.08.603566","DOIUrl":"https://doi.org/10.1101/2024.08.08.603566","url":null,"abstract":"Cotton fibers are aerial trichoblasts that employ a highly polarized diffuse growth mechanism to emerge from the developing ovule epidermis. After executing a complicated morphogenetic program, the cells reach lengths over 2 cm and serve as the foundation of a multi-billion-dollar textile industry. Important traits such as fiber diameter, length, and strength are defined by the growth patterns and cell wall properties of individual cells. At present, the ability to engineer fiber traits is limited by our lack of understanding regarding the primary controls governing the rate, duration, and patterns of cell growth. To gain insights into the compartmentalized functions of proteins in cotton fiber cells, we developed a label-free liquid chromatography mass spectrometry method for systems level analyses of fiber proteome. Purified fibers from a single locule were used to fractionate the fiber proteome into apoplast (APOT), membrane-associated (p200), and crude cytosolic (s200) fractions. Subsequently, proteins were identified, and their localizations and potential functions were analyzed using combinations of size exclusion chromatography, statistical and bioinformatic analyses. This method had good coverage of the p200 and apoplast fractions, the latter of which was dominated by proteins associated with particulate membrane-enclosed compartments. The apoplastic proteome was diverse, the proteins were not degraded, and some displayed distinct multimerization states compared to their cytosolic pool. This quantitative proteomic pipeline can be used to improve coverage and functional analyses of the cotton fiber proteome as a function of developmental time or differing genotypes.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941475","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.08.607179
Alexey Mikaberidze, Bruce A. McDonald, Lukas Kronenberg
Plants defend themselves against pathogens using either resistance, measured as the host's ability to limit pathogen multiplication, or tolerance, measured as the host's ability to reduce the negative effects of infection. Tolerance is a promising trait for crop breeding, but its genetic basis has rarely been studied and remains poorly understood. Here, we reveal the genetic basis of leaf tolerance to the fungal pathogen Zymoseptoria tritici that causes the globally important septoria tritici blotch disease on wheat. Leaf tolerance to Z. tritici is a quantitative trait that was recently discovered in wheat by using automated image analyses that quantified the symptomatic leaf area and counted the number of pycnidia found on the same leaf. A genome-wide association study including both tolerance and resistance to STB found a strong negative genetic correlation between these traits, indicative of a trade-off. We identified four chromosome intervals associated with tolerance and a separate chromosome interval associated with resistance. Within these intervals, we identified candidate genes, including wall-associated kinases similar to Stb6, the first cloned STB resistance gene. A trade-off between tolerance and resistance would hinder breeding simultaneously for both traits, but our findings suggest a way forward using marker-assisted breeding. We expect that the methods described here can be used to characterize tolerance to other fungal diseases that produce visible fruiting bodies, such as speckled leaf blotch on barley, potentially unveiling conserved tolerance mechanisms shared among plant species.
{"title":"A genome-wide association study identifies markers and candidate genes affecting tolerance to the wheat pathogen Zymoseptoria tritici","authors":"Alexey Mikaberidze, Bruce A. McDonald, Lukas Kronenberg","doi":"10.1101/2024.08.08.607179","DOIUrl":"https://doi.org/10.1101/2024.08.08.607179","url":null,"abstract":"Plants defend themselves against pathogens using either resistance, measured as the host's ability to limit pathogen multiplication, or tolerance, measured as the host's ability to reduce the negative effects of infection. Tolerance is a promising trait for crop breeding, but its genetic basis has rarely been studied and remains poorly understood. Here, we reveal the genetic basis of leaf tolerance to the fungal pathogen Zymoseptoria tritici that causes the globally important septoria tritici blotch disease on wheat. Leaf tolerance to Z. tritici is a quantitative trait that was recently discovered in wheat by using automated image analyses that quantified the symptomatic leaf area and counted the number of pycnidia found on the same leaf. A genome-wide association study including both tolerance and resistance to STB found a strong negative genetic correlation between these traits, indicative of a trade-off. We identified four chromosome intervals associated with tolerance and a separate chromosome interval associated with resistance. Within these intervals, we identified candidate genes, including wall-associated kinases similar to Stb6, the first cloned STB resistance gene. A trade-off between tolerance and resistance would hinder breeding simultaneously for both traits, but our findings suggest a way forward using marker-assisted breeding. We expect that the methods described here can be used to characterize tolerance to other fungal diseases that produce visible fruiting bodies, such as speckled leaf blotch on barley, potentially unveiling conserved tolerance mechanisms shared among plant species.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941476","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.09.607313
Sheila Rae E. Permanes, Youcef Mammeri, Melen Leclerc
Phenotyping host-pathogen interactions is crucial for understanding infectious diseases in plants. Traditionally, this process has relied on visual assessments or manual measurements, which can be subjective and labor-intensive. Recent advances in image processing and mathematical modeling enable precise and high-throughput phenotyping. In this study, we propose an innovative approach in plant pathology by combining image processing techniques with the level set method. This integrated approach leverages the strengths of both methodologies to provide accurate, robust, and detailed analysis of leaf and lesion evolution. By employing this combination, we achieve precise delineation of lesion boundaries and track their progression over time, offering clear visual feedback. This enhances the ability of the method to monitor plant health status comprehensively. The results, which track the growth of Peyronellaea pinodes on the stipules of two pea cultivars and the associated leaf deformation, provide an accurate visual representation of disease progression. This model represents a significant advancement in plant disease phenotyping, offering precise and detailed insights that can enhance our understanding of host-pathogen interactions.
{"title":"Spatio-temporal modeling of host-pathogen interactions using level set method","authors":"Sheila Rae E. Permanes, Youcef Mammeri, Melen Leclerc","doi":"10.1101/2024.08.09.607313","DOIUrl":"https://doi.org/10.1101/2024.08.09.607313","url":null,"abstract":"Phenotyping host-pathogen interactions is crucial for understanding infectious diseases in plants. Traditionally, this process has relied on visual assessments or manual measurements, which can be subjective and labor-intensive. Recent advances in image processing and mathematical modeling enable precise and high-throughput phenotyping. In this study, we propose an innovative approach in plant pathology by combining image processing techniques with the level set method. This integrated approach leverages the strengths of both methodologies to provide accurate, robust, and detailed analysis of leaf and lesion evolution. By employing this combination, we achieve precise delineation of lesion boundaries and track their progression over time, offering clear visual feedback. This enhances the ability of the method to monitor plant health status comprehensively.\u0000The results, which track the growth of Peyronellaea pinodes on the stipules of two pea cultivars and the associated leaf deformation, provide an accurate visual representation of disease progression. This model represents a significant advancement in plant disease phenotyping, offering precise and detailed insights that can enhance our understanding of host-pathogen interactions.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941368","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sr50, an intracellular nucleotide-binding leucine-rich repeat receptor (NLR), confers resistance of wheat against stem rust caused by the fungal pathogen Puccinia graminis f. sp. tritici. The receptor recognizes the pathogen effector AvrSr50 through its C-terminal leucine-rich repeat domain, initiating a localized cell death immune response. However, this immunity is compromised by mutations in the effector, as in the escape mutant AvrSr50QCMJC, which evades Sr50 detection. In this study, we employed iterative computational structural analyses and site-directed mutagenesis for rational engineering of Sr50 to gain recognition of AvrSr50QCMJC. Following an initial structural hypothesis driven by molecular docking, we identified the Sr50K711D single mutant, which induces an intermediate immune response against AvrSr50QCMJC without losing recognition against AvrSr50. Increasing gene expression with a stronger promoter enabled the mutant to elicit a robust response, indicating weak effector recognition can be complemented by enhanced receptor expression. Further structural refinements led to the creation of five double mutants and two triple mutants with dual recognition of AvrSr50 and AvrSr50QCMJC with greater immune response intensities than Sr50K711D against the escape mutant. All effective mutations against AvrSr50QCMJC required the K711D substitution, indicating that multiple solutions exist for gain of recognition, but the path to reach these mutations may be confined. Furthermore, this single substitution alters the prediction of AlphaFold 2, allowing it to model the complex structure of Sr50K711D and AvrSr50 that match our final structural hypothesis. Collectively, our study outlines a framework for rational engineering of NLR systems to overcome pathogen escape mutations and provides datasets for future computational models for NLR resurrection.
{"title":"Engineering the plant intracellular immune receptor Sr50 to restore recognition of the AvrSr50 escape mutant","authors":"Kyungyong Seong, Wei Wei, Brandon Vega, Amanda Dee, Griselda Ramirez-Bernardino, Rakesh Kumar, Lorena Parra, Ksenia Krasileva","doi":"10.1101/2024.08.07.607039","DOIUrl":"https://doi.org/10.1101/2024.08.07.607039","url":null,"abstract":"Sr50, an intracellular nucleotide-binding leucine-rich repeat receptor (NLR), confers resistance of wheat against stem rust caused by the fungal pathogen <em>Puccinia graminis</em> f. sp. <em>tritici</em>. The receptor recognizes the pathogen effector AvrSr50 through its C-terminal leucine-rich repeat domain, initiating a localized cell death immune response. However, this immunity is compromised by mutations in the effector, as in the escape mutant AvrSr50<sup>QCMJC</sup>, which evades Sr50 detection. In this study, we employed iterative computational structural analyses and site-directed mutagenesis for rational engineering of Sr50 to gain recognition of AvrSr50<sup>QCMJC</sup>. Following an initial structural hypothesis driven by molecular docking, we identified the Sr50<sup>K711D</sup> single mutant, which induces an intermediate immune response against AvrSr50<sup>QCMJC</sup> without losing recognition against AvrSr50. Increasing gene expression with a stronger promoter enabled the mutant to elicit a robust response, indicating weak effector recognition can be complemented by enhanced receptor expression. Further structural refinements led to the creation of five double mutants and two triple mutants with dual recognition of AvrSr50 and AvrSr50<sup>QCMJC</sup> with greater immune response intensities than Sr50<sup>K711D</sup> against the escape mutant. All effective mutations against AvrSr50<sup>QCMJC</sup> required the K711D substitution, indicating that multiple solutions exist for gain of recognition, but the path to reach these mutations may be confined. Furthermore, this single substitution alters the prediction of AlphaFold 2, allowing it to model the complex structure of Sr50<sup>K711D</sup> and AvrSr50 that match our final structural hypothesis. Collectively, our study outlines a framework for rational engineering of NLR systems to overcome pathogen escape mutations and provides datasets for future computational models for NLR resurrection.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-08DOI: 10.1101/2024.08.07.607067
Yezhang Ding, Hunter K. Vogel, Yi Zhai, Hans K. Carlson, Peter F. Andeer, Vlastimil Novak, Nakian Kim, Benjamin P. Bowen, Amber N. Golini, Suzanne M. Kosina, Devin Coleman-Derr, John P. Vogel, Trent R. Northen
Dopamine plays a critical role in animal physiology and interactions with gut microbes. In plants, dopamine is known to function in plant defense and abiotic stress tolerance; however, its role in mediating plant-microbiome interactions remains unexplored. In this study, we observed that dopamine is one of the most abundant exometabolites with natural variation in root exudates across diverse Brachypodium distachyon lines, suggesting a potential role in rhizosphere microbial assembly. To further investigate this, we colonized ten natural B. distachyon lines with a 16-member bacterial synthetic community (SynCom), collected paired metabolomic and 16S rRNA sequencing data, and performed an association analysis. Our results revealed that dopamine levels in root exudates were significantly associated with the abundance of six SynCom members in a hydroponic system. In vitro growth studies demonstrated that dopamine had a significant effect on the growth of the same six bacterial isolates. Additionally, treating soil directly with dopamine enriched Actinobacteria, consistent with both the SynCom-dopamine correlations and the isolate growth results. Collectively, our study underscores the selective influence of dopamine on rhizosphere microbial communities, with implications for precision microbiome management.
{"title":"Insights into the role of dopamine in rhizosphere microbiome assembly","authors":"Yezhang Ding, Hunter K. Vogel, Yi Zhai, Hans K. Carlson, Peter F. Andeer, Vlastimil Novak, Nakian Kim, Benjamin P. Bowen, Amber N. Golini, Suzanne M. Kosina, Devin Coleman-Derr, John P. Vogel, Trent R. Northen","doi":"10.1101/2024.08.07.607067","DOIUrl":"https://doi.org/10.1101/2024.08.07.607067","url":null,"abstract":"Dopamine plays a critical role in animal physiology and interactions with gut microbes. In plants, dopamine is known to function in plant defense and abiotic stress tolerance; however, its role in mediating plant-microbiome interactions remains unexplored. In this study, we observed that dopamine is one of the most abundant exometabolites with natural variation in root exudates across diverse Brachypodium distachyon lines, suggesting a potential role in rhizosphere microbial assembly. To further investigate this, we colonized ten natural B. distachyon lines with a 16-member bacterial synthetic community (SynCom), collected paired metabolomic and 16S rRNA sequencing data, and performed an association analysis. Our results revealed that dopamine levels in root exudates were significantly associated with the abundance of six SynCom members in a hydroponic system. In vitro growth studies demonstrated that dopamine had a significant effect on the growth of the same six bacterial isolates. Additionally, treating soil directly with dopamine enriched Actinobacteria, consistent with both the SynCom-dopamine correlations and the isolate growth results. Collectively, our study underscores the selective influence of dopamine on rhizosphere microbial communities, with implications for precision microbiome management.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941346","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-07DOI: 10.1101/2024.08.04.606528
Vincent J. Pennetti, Peter R. LaFayette, Wayne Allen Parrott
Agrobacterium mediated plant transformation largely depends on two distinct strain lineages – C58 and Ach5. To better serve the plant transformation community, we have created a suite of auxotrophic and auxotrophic recombinant deficient mutants of C58 derivatives EHA105, GV3101::pMP90, and Ach5 derivative LBA4404. While these derivatives are useful, having additional strain backgrounds available would help expand the repertoire for plant transformation even further. Toward that end, two underutilized hypervirulent strains are K599 (NCPPB 2659), and Chry5—but disarmed variants are not easily accessible. To improve availability, we produced disarmed versions of A. rhizogenes strain K599 and A. tumefaciens strain Chry5 and introduced the same desirable mutations as with the other lineages. Each thymidine auxotrophy and recombination deficiency were introduced to existing and newly disarmed Agrobacterium strains via loss of function mutations conferred to thyA and recA, respectively, through CRISPR-mediated base-editing of codons amenable to nonsense mutation. To streamline the editing process, we created a series of visually marked single component base-editor vectors and a corresponding guide-filtering Geneious Prime wrapper plugin for expedited guide filtering. These new strains, the simplified CRISPR-mediated base-editor plasmids, and streamlined workflow will improve the ease with which future Agrobacterium strain derivatives are created while also supporting plant transformation at large.
{"title":"Single component CRISPR-mediated base- editors for Agrobacterium and their use to develop an improved suite of strains","authors":"Vincent J. Pennetti, Peter R. LaFayette, Wayne Allen Parrott","doi":"10.1101/2024.08.04.606528","DOIUrl":"https://doi.org/10.1101/2024.08.04.606528","url":null,"abstract":"Agrobacterium mediated plant transformation largely depends on two distinct strain lineages – C58 and Ach5. To better serve the plant transformation community, we have created a suite of auxotrophic and auxotrophic recombinant deficient mutants of C58 derivatives EHA105, GV3101::pMP90, and Ach5 derivative LBA4404. While these derivatives are useful, having additional strain backgrounds available would help expand the repertoire for plant transformation even further. Toward that end, two underutilized hypervirulent strains are K599 (NCPPB 2659), and Chry5—but disarmed variants are not easily accessible. To improve availability, we produced disarmed versions of <em>A. rhizogenes</em> strain K599 and <em>A. tumefaciens</em> strain Chry5 and introduced the same desirable mutations as with the other lineages. Each thymidine auxotrophy and recombination deficiency were introduced to existing and newly disarmed Agrobacterium strains via loss of function mutations conferred to <em>thyA</em> and <em>recA</em>, respectively, through CRISPR-mediated base-editing of codons amenable to nonsense mutation. To streamline the editing process, we created a series of visually marked single component base-editor vectors and a corresponding guide-filtering Geneious Prime wrapper plugin for expedited guide filtering. These new strains, the simplified CRISPR-mediated base-editor plasmids, and streamlined workflow will improve the ease with which future <em>Agrobacterium</em> strain derivatives are created while also supporting plant transformation at large.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941481","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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