Progress 11/22/16 to 09/30/21
Outputs
Target Audience: 1. Researchers in universities and biotechnology companies interested in the biology of oomycete plant pathogens and strategies for controlling their diseases. 2. Students seeking training in oomycete biology and molecular plant pathology. Changes/Problems:Progress in several areas was delayed by difficulties in establishing an efficient gene editing system for P. infestans. The CRISPR/Cas9 system commonly used in other species proved to be toxic to P. infestans. We obtained better results using Cas12a, an alternate gene editing protein. While the method still needs to be optimized, we have set the groundwork for improved success in studies of gene function in oomycetes. What opportunities for training and professional development has the project provided? Junior scientists (graduate students) were trained in molecular biology, genomics, cell biology, and plant pathology. How have the results been disseminated to communities of interest? Our group published referreed papers in scientific journals during the past year, most in open-access journals. We also maintained our web site, which disseminates information and the latest protocols on oomycete transformation. We alsodistributed transformation plasmids to 12 labs in Asia and Europe. We have also consulted with several biotechnology companies interested in identifying fungicide targets in oomycetes. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Overall impact of our research:The overall objective of the research is to improve our understanding of the biology of oomycete plant pathogens such as P. infestans, with the longer-term goal of identifying factors that might serve as targets of crop protection chemicals. Towards those ends, we identified several proteins that regulate pathways needed for disseminating the pathogen from field to field (e.g. the production of wind-blown sporangia) and for plant infection (e.g. the germination of those sporangia). As part of this research, we also developed and tested new technologies for mutating genes, which is an important tool needed to test whether a particular protein plays an important role. We also shed light on what causes variation between strains of P. infestans; prior studies have revealed that strains may differ in important characteristics such as sporulation intensity, fungicide resistance, and pathogenic aggressiveness. We found that factors underlying these differences include aberrations in the chromosomes, such as deletions of segments of the chromosomes or changes in chromosome number. Objective 1: characterizing genes and proteins involved in sporulation and plant infection:To understand what regulates gene expression during the life cycle, we used methods such as protein binding arrays, electrophoretic mobility shift assays, and yeast one-hybrid to identify and validate the binding specificity of approximately 70 P. infestans transcription factors. We also used a novel bioinformatic approach to link these binding specificities to patterns of gene expression during the life cycle. These results were validated in several cases using reporter gene assays, in which promoters containing the motif of interest (or versions in which the motifs were mutated) were fused to the GUS reporter gene and expressed in stable transformants of P. infestans. Other studies characterized a regulatory module involved in regulating sporulation in P. infestans. Using a range of experimental methods for studying protein-protein interactions such as co-immunoprecipitation assays and yeast two-hybrid, binding was detected between a transcription factor, a protein kinase, and a protein phosphatase. Based on studies of phosphorylation it appears that the kinase may phosphorylate the transcription factor, while later in the sporulation cycle this modification is reversed by the phosphatase. The latter appears to destabilize the transcription factor as a necessary step in progression through the life cycle. Silencing or gene editing using Cas12a showed that the transcription factor and protein phosphatase are essential for sporulation. A putative co-regulator of the transcription factor was also characterized. Using confocal microscopy using fluorescent tags, this was shown to accumulate in nuclei, particularly during sporulation. Gene editing has suggested that this regulator may control spore germination. In support of these and other studies, to improve genomic tools for manipulating genes, we continued our development of a new gene editing tool for P. infestans using the Cas12a editing nuclease. This was used in two applications. First, it was used to edit and then block the expression of a protein kinase proved to be involved in sporulation. Second, the technology was used to tag native genes with fluorescent proteins, for application in subcellular localization studies. Objective 3: Studies of the molecular basis of fungicide resistance and modes of action:Many studies in the literature have indicated that continued applications of fungicides can induce higher levels of resistance to the fungicide in the pathogen. Using the crop protection chemical metalaxyl, we showed that chromosome abnormalities are often associated with an increase in resistance in P. infestans. Further analysis mapped a locus determining resistance to the arm of one particular chromosome. Moreover, continued growth in the presence of metalaxyl selected for strains in which one arm of that chromosome was deleted, effectively reducing the copy number of the gene implicating as being the target of the chemical. To explore new methods for inhibiting oomycetes, we tested the ability of exogenously added double-stranded RNA molecules to be taken up by Phytophthora infestans; similar technologies have been used experimentally against fungi for controlling their plant diseases. Unfortunately, most life stages of P. infestans poorly took up the RNA molecules. suggesting that this technology can not be applied to at least some oomycetes. Objective 4: Gene and genome structure in oomycetes:Studies of the effect of polyploidy on P. infestans were initiated. Polyploid and aneuploid progeny were found to be produced at an unusually high frequency during the sexual reproduction of diploids. Aneuploidy was found to result in abnormally imbalanced patterns of transcription, based on RNA-seq comparisons of diploid and aneuploid offspring. However, aneuploidy did not seem to have a major effect on growth orpathogenicity. Studies were also initiated to study the basis of expression level polymorphisms between strains of P. infestans. Besides quantitative differences resulting from chromosome imbalances, events such as point mutations, genome rearrangements including inversions and deletions, and transposable element insertions were common factors underlying expression polymorphisms. Other expression differences could not be linked to a stable DNA modification, suggesting that epigenetic factors might be involved.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Qiao L, Lan C, Capriotti L, Ah-Fong A, Nino Sanchez J, Hamby R, Heller J, Zhao H, Glass NL, Judelson HS, Mezzetti B, Niu D, Jin H. Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake. Plant Biotechnol J. 2021 Sep;19(9):1756-1768
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Stajich JE, Vu AL, Judelson HS, Vogel GM, Gore MA, Carlson MO, Devitt N, Jacobi J, Mudge J, Lamour KH, Smart CD. High-Quality Reference Genome Sequence for the Oomycete Vegetable Pathogen Phytophthora capsici Strain LT1534. Microbiol Resour Announc. 2021 May 27;10(21):e0029521
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Ah-Fong AMV, Boyd AM, Matson MEH, Judelson HS. A Cas12a-based gene editing system for Phytophthora infestans reveals monoallelic expression of an elicitor. Mol Plant Pathol. 2021 Jun;22(6):737-752
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Progress 10/01/19 to 09/30/20
Outputs
Target Audience: 1. Researchers in universities and biotechnology companies interested in the biology of oomycete plant pathogens and strategies for controlling their diseases. 2. Students seeking training in oomycete biology and molecular plant pathology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Junior scientists (graduate students and a postdoctoral) were trained in molecular biology, genomics, cell biology, and plant pathology. How have the results been disseminated to communities of interest? Our group published referreed papers in scientific journals during the past year, most in open-access journals.We also maintained our web site, which disseminates information and the latest protocols on oomycete transformation, and distributed transformation plasmids to about 5 labs in Asiaand Europe. What do you plan to do during the next reporting period to accomplish the goals? 1. Optimize thenew gene editing methods for gene function analysis in P. infestans. 2. Test the function of transcription factors involved in expressing genes during sporulation, spore germination, and plant infection. 3. Identify protein interactors of transcription factors and environment-sensing proteins that are believed to be involved in sporulation.
Impacts
What was accomplished under these goals?
1. To understand howPhytophthora infestansfeeds from host plants, we studied its two extracellular invertases. Unlike typical fungal invertases, theP. infestansgenes are not sucrose induced or glucose repressed but instead appear to be under developmental control. Transcript levels of both genes were very low in mycelia harvested from artificial medium but high in preinfection stages (sporangia, zoospores, and germinated cysts), high during biotrophic growth in leaves and tubers, and low during necrotrophy. Genome-wide analyses of metabolic enzymes and effectors indicated that this expression profile was fairly unusual, matched only by a few other enzymes, such as carbonic anhydrases and a few RXLR effectors. Genes for other metabolic enzymes were typically downregulated in the preinfection stages. Overall metabolic gene expression during the necrotrophic stage of infection clustered with artificial medium, while the biotrophic phase formed a separate cluster. Confocal microscopy of transformants expressing green fluorescent protein (GFP) fusions indicated that invertase protein resided primarily in haustoria during infection. This localization was not attributable to haustorium-specific promoter activity. Instead, the N-terminal regions of proteins containing signal peptides were sufficient to deliver proteins to haustoria. Invertase expression during leaf infection was linked to a decline in apoplastic sucrose, consistent with a role of the enzymes in plant pathogenesis. This was also suggested by the discovery that invertase genes occur across multiple orders of oomycetes but not in most animal pathogens or a mycoparasite. 2. In another study related to metabolism, we characterized nutrient utilization during the growth of P. infestans on artificial media and in plant apoplastic fluids. Biased use of nitrogen compounds were observed in both cases, with preferential nitrogen sources identified. The usage patterns of several of these were implicated as factors regulating the transition from biotrophy to necrotrophy and sporulation. 3. To understand what regulates gene expression during plant infection, we identified promoter motifs driving effector expression using a variety of bioinformatic and functional genomics assays. The activity of the cognate transcription factors during stages of the life cycle were determined and integrated with studies of metabolic changes during growth and development. 4. We also contributed to projects aimed at understanding the population genetics of P. infestans by using a new chromosome-scale genome assembly as a reference for comparisons.This involved a comparative genomic analysis of 47 high-coverage genomes to infer changes in genic copy number. Conclusions included that sexual populations at the center of origin are diploid, as was the lineage that caused the famine, while modern clonal lineages showed increased copy number, typically triploidy. Copy number variation (CNV) was found genome-wide. It is theorized that a higher copy number provides fitness, leading to replacement of prior clonal lineages. 5. We also expanded our studies to other oomycetes to help understand the diversity within the group. Using a new assembly of the basil downy mildew pathogenPeronosopora belbahrii, we examined components of the genome including promoter stucture. Compared to other oomycetes including P. infestans, the base composition bias and microsatellite components of intergenic were substantially different. Moreover, most promoter motifs identified in other species showed variation in sequence. 6. In a related comparative genomics project, we developed an improved assembly of Phytophthora capsici, which is mostly a pathogen of vegetables. The assembly was used to identify gene sequences which were used for transcriptomic studies of sporulation, including the effects of environmental factors on sporulation. 7. To improve genomic tools for manipulating genes, we developed a new gene editing tool for P. infestans using the Cas12a editing nuclease. This represents the first successful editing technology for P. infestans and should increase the pace of functional genomics studies in the species.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Dov Prusky, Leandro Jos� De Assis, Riccardo Baroncelli, Ernesto P Benito, Virginia Casado del Castillo, Timothy Chaya, Shay Covo, Jos� Mar�a D�az-M�nguez, Nicole M Donofrio, Eduardo Espeso, T�nia Ribeiro Fernandes, Gustavo H Goldman, Howard Judelson, Daniela Nordzieke, Antonio Di Pietro, Edward Sionov, Serenella A Sukno, Michael R Thon, Richard B Todd, Lars Voll, Jin Rong Xu, Benjamin A Horwitz, Richard A Wilson (2020) Nutritional factors modulating plant and fruit susceptibility to pathogens. Phytoparasitica 48: 317-333
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Brian J Knaus, Javier F Tabima, Shankar K Shakya, Howard S Judelson, Niklaus J Gr�nwald (2020) Genome-wide increased copy number is associated with emergence of dominant clones of the Irish potato famine pathogen Phytophthora infestans. MBio 11:e00326-20
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Marco Thines, Rahul Sharma, Sander YA Rodenburg, Anna Gogleva, Howard S Judelson, Xiaojuan Xia, Johan van den Hoogen, Miloslav Kitner, Jo�l Klein, Manon Neilen, Dick de Ridder, Michael F Seidl, Guido Van den Ackerveken, Francine Govers, Sebastian Schornack, David J Studholme (2020). The Genome of Peronospora belbahrii Reveals High Heterozygosity, a Low Number of Canonical Effectors, and TC-Rich Promoters. Molecular Plant-Microbe Interactions 33: 742-753.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Meenakshi S Kagda, Domingo Mart�nez-Soto, Audrey MV Ah-Fong, Howard S Judelson. (2020). Invertases in Phytophthora infestans Localize to Haustoria and Are Programmed for Infection-Specific Expression. mBio. 11: e01251-20
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience: 1. Researchers in universities and biotechnology companies interested in the biology of oomycete plant pathogens and strategies for controlling their diseases. 2. Students seeking training in oomycete biology and molecular plant pathology. Changes/Problems:We consider the development of gene editing tools for P. infestans to be a high priority. However, the apparent toxicityof Cas9 has caused us to shift efforts to other editing proteins. What opportunities for training and professional development has the project provided?Junior scientists (graduate students and a postdoctoral)were trained in molecular biology, genomics, cell biology, and plant pathology. This included hosting an undergraduate researcher from the United States, a visiting graduate student from The Netherlands, and a visiting scientist from Japan. How have the results been disseminated to communities of interest? Our group published referreed papers in scientific journals during the past year, most in open-access journals. In addition, presentations were made at the Annual Oomycete Molecular Genetics Workshop and the Plant and Animal Genome Conference. We also maintained our web site, which disseminates information and the latest protocols on oomycete transformation, and distributed transformation plasmids to about 12 labs in Asia, North America, and Europe. What do you plan to do during the next reporting period to accomplish the goals? 1. Test new gene editing methods for gene function analysis in P. infestans and relatives, which will help in testing gene function. 2. Test the function of transcription factors involved in expressing genes during sporulation, spore germination, and plant infection. 3. Study the effect of metabolite levels and light on sporulation. 4. Identify protein interactors of transcription factors and light-sensing proteins that are believed to be involved in sporulation.
Impacts
What was accomplished under these goals?
1. We addressed the relationship between metabolism and trophic behavior by comparing metabolic gene expression during potato tuber colonization by two oomycetes, the hemibiotrophPhytophthora infestansand the necrotrophPythium ultimum. Genes for several pathways including amino acid, nucleotide, and cofactor biosynthesis were expressed more byPh.infestansduring its biotrophic stage compared toPy.ultimum. In contrast,Py.ultimumhad higher expression of genes for metabolizing compounds that are normally sequestered within plant cells but released to the pathogen upon plant cell lysis, such as starch and triacylglycerides. The transcription pattern of metabolic genes inPh.infestansduring late infection became more like that ofPy.ultimum, consistent with the former's transition to necrotrophy. Interspecific variation in metabolic gene content was limited but included the presence of γ-amylase only inPy.ultimum. The pathogens were also found to employ strikingly distinct strategies for using nitrate. Measurements of mRNA,15N labeling studies, enzyme assays, and immunoblotting indicated that the assimilation pathway inPh.infestanswas nitrate-insensitive but induced during amino acid and ammonium starvation. In contrast, the pathway was nitrate-induced but not amino acid-repressed inPy.ultimum. The lack of amino acid repression inPy.ultimumappears due to the absence of a transcription factor common to fungi andPhytophthorathat acts as a nitrogen metabolite repressor. Evidence for functional diversification in nitrate reductase protein was also observed. Its temperature optimum was adapted to each organism's growth range, and its Kmwas much lower inPy.ultimum. In summary, we observed divergence in patterns of gene expression, gene content, and enzyme function which contributed to the fitness of each species in its niche. 2. To improve our understanding of DNA transformation and homology-based transcriptional silencing, we usedP. infestansto study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spread to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5' ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for 12 different gene targets indicated that neighbors within 500 nt were often cosilenced, regardless of whether hairpin or sense constructs were employed and the direction of transcription of the target. However, thiscisspreading of silencing did not occur in all transformants obtained with the same plasmid. Genome-wide studies indicated that unlinked genes with partial complementarity with the silencing-inducing transgene were not usually down-regulated. We learned that hairpin or sense transgenes were not cosilenced with the target in all transformants, which informs how screens for silencing should be performed. We concluded that transformation and gene silencing can be reliable tools for functional genomics inPhytophthoraspp. but must be used carefully, especially by testing for the spread of silencing to genes flanking the target. 3. To refine our understanding of sporulation, we investigated whether sporangia from artificial media and plant lesions are functionally equivalent. We compared the transcriptomes and infection ability of sporangia from rye-sucrose media, potato and tomato leaflets, and potato tubers. Small differences were observed between the mRNA profiles of sporangia from all sources, including variation in genes encoding metabolic enzymes, cell-wall-degrading enzymes, and ABC transporters. Small differences in sporangia age also resulted in variation in the transcriptome. Taking care to use sporangia of similar maturity, we observed that those sourced from media or plant lesions had similar rates of zoospore release and cyst germination. There were also no differences in infection rates or aggressiveness on leaflets, based on single-spore inoculation assays. Such results are discordant with those of a recent publication in this journal. Nevertheless, we conclude that sporangia from plant and media cultures are functionally similar and emphasized the importance of using "best practices" with sporangia to obtain reliable results. 4. To understand energy use in the flagellated spores ofP. infestans, we found that a dimeric PK is found at low levels in vegetative mycelia, but high levels in ungerminated sporangia and zoospores. In contrast, a monomeric PK protein was at similar levels in all tissues, although being transcribed primarily in mycelia. Subcellular localization studies indicated that the monomeric PK is mitochondrial. In contrast, the dimeric PK was cytoplasmic in mycelia and sporangia but retargeted to flagellar axonemes during zoosporogenesis. This supports a model in which PKs shuttle energy from mitochondria to and through flagella. Metabolite analysis indicates that deployment of the flagellar PK is coordinated with a large increase in taurocyamine, synthesized by sporulation-induced enzymes that were lost during the evolution of zoospore-lacking oomycetes. Thus, PK function is enabled by coordination of the transcriptional, metabolic and protein targeting machinery during the life cycle. 5. To help understand how genes are regulated during growth and development, we identified the binding specificity of over 70P. infestanstranscription factors using protein binding arrays. Candidates for regulators of particular developmental stages were identified by determining whether each motif was over-represented in the promoters of genes up-regulated at particular stages of development. 6. To bring gene editing technologies to P. infestans, we tested the expression of catalytically active and dead versions of the Cas9 and Cas12a CRISPR proteins. Conclusions were drawn that suggested that some of the enzymes were lethal if consitutively expressed. Different nuclear localization signals were tested for efficacy.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
SYA Rodenburg, MF Seidl, HS Judelson, AL Vu, F Govers, D de Ridder. 2019. Metabolic model of the Phytophthora infestans-tomato interaction reveals metabolic switches during host colonization. mBio 10 (4), e00454-19
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
AMV Ah-Fong, MS Kagda, M Abrahamian, HS Judelson. 2019. Niche-specific metabolic adaptation in biotrophic and necrotrophic oomycetes is manifested in differential use of nutrients, variation in gene content, and enzyme evolution. PLoS pathogens 15 (4), e1007729
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
HS Judelson, AMV Ah-Fong. 2019. Exchanges at the plant-oomycete interface that influence disease. Plant physiology 179 (4), 1198.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
W Leesutthiphonchai, H Judelson. 2019. Phytophthora infestans sporangia produced in artificial media and plant lesions have subtly divergent transcription profiles but equivalent infection potential and aggressiveness. Molecular Plant Microbe Interactions 32: 1077 -1087
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
AL Vu, W Leesutthiphonchai, AMV Ah-Fong, HS Judelson. 2019. Defining transgene insertion sites and off-target effects of homology-based gene silencing informs the use of functional genomics tools in Phytophthora infestans. Molecular Plant Microbe Interactions 32: 915927.
& - Molecular Plant-Microbe Interactions, 2019
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
MS Kagda, AL Vu, AMV Ah Fong, HS Judelson. 2018. Phosphagen kinase function in flagellated spores of the oomycete Phytophthora infestans integrates transcriptional regulation, metabolic dynamics and protein targeting. Molecular microbiology 110 (2), 296-308
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience: 1. Researchers in universities and biotechnology companies interested in the biology of oomycete plant pathogens and strategies for controlling their diseases. 2. Students seeking training in oomycete biology and molecular plant pathology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Junior scientists were trained in molecular biology, genomics, cell biology, and plant pathology. This included hosting an undergraduate researcher. How have the results been disseminated to communities of interest? Our group published referreed papers in scientific journals during the past year, plus a review and book chapter. In addition, presentations were made at the Annual Meeting of the Korean Society of Plant Pathology, and at the European Fungal Genetics Conference.. We also maintained our web site which disseminates information and the latest protocols on oomycete transformation, and distributed transformation plasmids to about 10 labs in Asia, North America, and Europe. What do you plan to do during the next reporting period to accomplish the goals? 1. Localize metabolic enzymes and nutrient transporters that are believed to contribute to fitness and pathogenicity. 2. Conduct functional testing of selected metabolic enzymes and nutrient transporters. 3. Test new gene editingmethods for gene function analysis in P. infestans and relatives, which will help in testing gene function. 4. Assess the function of genes believed to determinenecrotrophy, and regulatory proteins that are believed to regulate sporulation.
Impacts
What was accomplished under these goals?
1. To understand what regulates asexual sporulation, we studied a MADS-box transcription factor. We found that PiMADS is not transcribed during vegetative growth, but is induced early during asexual sporulation. Its mRNA levels oscillated in response to light, which suppresses sporulation. The protein was not detected in nonsporulating mycelia, but was found in sporulating mycelia and spores. Both mRNA and protein levels decline upon spore germination. A similar expression pattern as well as nuclear localization was observed when the protein was expressed with a fluorescent tag from the native promoter. Gene silencing indicated that PiMADS is required for sporulation but not hyphal growth or plant colonization. A comparison of wild type to a silenced strain by RNA-seq indicated that PiMADS regulates about 3000 sporulation-associated genes, and acts before other genes previously shown to regulate sporulation. Analysis of the silenced strain also indicated that the native gene was not transcribed while the transgene was still expressed, which contradicts current models for homology-dependent silencing in oomycetes. 2. To expand our knowledge of sexual spore production, we used RNA-seq and quantitative proteomics to examine matings inP. infestans. Exhibiting significant changes in mRNA abundance during matings were 1170 genes, most being mating-induced. Most of these genes had elevated expression in a self-fertile strain. Many were associated with cell wall biosynthesis, which may relate to forming the thick-walled sexual spore. Several gene families were induced during mating including one encoding histidine, serine, and tyrosine-rich putative wall proteins, and another encoding prolyl hydroxylases which may strengthen the extracellular matrix. Proteomic analyses of mature oospores and nonmating hyphae using isobaric tags for quantification identified 835 shared proteins, with 5% showing>2-fold changes in abundance between the tissues. Enriched in oospores wereβ-glucanases potentially involved in digesting the oospore wall during germination. Despite being dormant, oospores contained a mostly normal complement of proteins required for core cellular functions. 3.We improved methods for studying gene expression in oomycetes by identifying new housekeeping controls that are more stable thanexisting normalization standards.We also observed >2-fold variation in the fraction of polyA+RNA between life stages, which suggest that a form oftranslational control may be important during sexual development. 4. We improved the annotation of the P. infestans genome by using RNA-seq data along with the MAKER program to identify genes in a new assembly of the genome that is based on long-read technology. 5. We explored a potentialtarget for future fungicides by studyingphosphagen kinases (PKs). We found that P. infestans expresses adimeric PK iat low levels in vegetative mycelia, but high levels in ungerminated sporangia and zoospores. In contrast, a monomeric PK protein was at similar levels in all tissues, although is transcribed primarily in mycelia. Subcellular localization studies indicated that the monomeric PK is mitochondrial. In contrast, the dimeric PK is cytoplasmic in mycelia and sporangia but is retargeted to flagellar axonemes during zoosporogenesis. This supports a model in which PKs shuttle energy from mitochondria to and through flagella. Metabolite analysis indicates that deployment of the flagellar PK is coordinated with a large increase in taurocyamine, synthesized by sporulation?induced enzymes that were lost during the evolution of zoospore?lacking oomycetes. Thus, PK function appears to be enabled by coordination of the transcriptional, metabolic and protein targeting machinery during the life cycle. Since plants lack PKs, the enzymes may be useful targets for inhibitors of oomycete plant pathogens.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Leesutthiphonchai W, Vu AL, Ah-Fong AMV, Judelson HS. 2018.
Phytopathology. 2018. How Does Phytophthora infestans Evade Control Efforts? Modern Insight Into the Late Blight Disease. 108:916-924.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Leesutthiphonchai W, Judelson HS. 2018. A MADS?box transcription factor regulates a central step in sporulation of the oomycete Phytophthora infestans. Molecular Microbiology 110: 562-575
- Type:
Book Chapters
Status:
Published
Year Published:
2018
Citation:
Ah-Fong A, Kagda M, Judelson HS. 2018. Illuminating Phytophthora Biology with Fluorescent Protein Tags. Methods in Molecular Biology 1848:119-129
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Niu X, Ah-Fong AMV, Lopez L, Judelson HS. 2018. Transcriptomic and proteomic analysis reveals wall-associated and glucan-degrading proteins with potential roles in Phytophthora infestans sexual spore development. PLOS One 13:e0198186
|
Progress 11/22/16 to 09/30/17
Outputs
Target Audience:1. Researchers in universities and biotechnology companies interested in the biology of oomycete plant pathogens and strategies for controlling their diseases. 2. Students seeking training in oomycete biology and molecular plant pathology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Junior scientists were trained in molecular biology, genomics, cell biology, and plant pathology. This included hosting an undergraduate researcher. How have the results been disseminated to communities of interest?Our group published referreed papers in scientific journals during the past year, plus a review. In addition, presentations were madeat the Oomycete Molecular Genetics Conference. We also maintained our web site which disseminates information and the latest protocols on oomycete transformation, and distributed transformation plasmids to >10 labs in Asia, North America, and Europe. What do you plan to do during the next reporting period to accomplish the goals?1. Localize metabolic enzymes and nutrient transporters that are believed to contribute to fitness and pathogenicity. 2. Conduct a detailed comparison of the timing of expression of such genes in P. infestans and Py. ultimum. 3. Conduct functional testing of selected metabolic enzymes and nutrient transporters. 4. Test new methods for gene function analysis in P. infestans, to help accelerate goal #3. 5. Assess the function of genes believed to determine biotrophy and necrotrophy.
Impacts
What was accomplished under these goals?
To help learn how phytopathogens feed, genes for nutrient transporters from the hemibiotrophic potato and tomato pest Phytophthora infestans were annotated. This identified 453 genes from 19 families. Comparisons with a necrotrophic oomycete, Pythium ultimum var. ultimum, and a hemibiotrophic fungus, Magnaporthe oryzae, revealed diversity in the size of some families although a similar fraction of genes encoded transporters. RNA-seq of infected potato tubers, tomato leaves, and several artificial media revealed that 56 and 207 transporters from P. infestans were significantly up- or down-regulated, respectively, during early infection timepoints of leaves or tubers versus media. About 17 were up-regulated >4-fold in both leaves and tubers compared to media and expressed primarily in the biotrophic stage. The transcription pattern of many genes was host-organ specific. For example, the mRNA level of a nitrate transporter (NRT) was about 100-fold higher during mid-infection in leaves, which are nitrate-rich, than in tubers and three types of artificial media, which are nitrate-poor. The NRT gene is physically linked with genes encoding nitrate reductase (NR) and nitrite reductase (NiR), which mobilize nitrate into ammonium and amino acids. All three genes were coregulated. For example, the three genes were expressed primarily at mid-stage infection timepoints in both potato and tomato leaves, but showed little expression in potato tubers. Transformants down-regulated for all three genes were generated by DNA-directed RNAi, with silencing spreading from the NR target to the flanking NRT and NiR genes. The silenced strains were nonpathogenic on leaves but colonized tubers. We propose that the nitrate assimilation genes play roles both in obtaining nitrogen for amino acid biosynthesis and protecting P. infestans from natural or fertilization-induced nitrate and nitrite toxicity. To learn how pathogen genomes evolved to support distinct lifestyles, we compared the hemibiotroph P. infestans with Py. ultimum in media and on a shared host, potato tuber. Genes related to pathogenesis varied in temporal expression pattern, mRNA level, and family size between the species. A family's aggregate expression during infection was not proportional to size due to transcriptional remodeling and pseudogenization. P. infestans had more stage-specific genes, while Py. ultimum tended towards more constitutive expression. P. infestans expressed more genes encoding secreted cell wall-degrading enzymes, but other categories such as secreted proteases and ABC transporters had higher transcript levels in Py. ultimum. Species-specific genes were identified including new Pythium genes, perforins, which may disrupt plant membranes. Genome-wide ortholog analyses identified substantial diversified expression, which correlated with sequence divergence. Pseudogenization was associated with gene family expansion, especially in gene clusters. Overall, the data suggest that biotrophy and necrotrophy are determined by species-specific genes and the varied expression of shared pathogenicity factors. To place the observed expression patterns of P. infestans genes in planta in context with the life cycle, RNA-seq was used to profile hyphae, sporangia, sporangia undergoing zoosporogenesis, motile zoospores, and germinated cysts. Parallel studies of two isolates generated robust expression calls for 16,000 of 17,797 predicted genes. The largest changes occurred in the transition from hyphae to sporangia, when >4200 genes were up-regulated. Proteins associated with pathogenicity were transcribed in waves with subclasses induced during zoosporogenesis, in zoospores, or in germinated cysts. Most genes in metabolic pathways were down-regulated upon sporulation and reactivated during cyst germination. Inhibitor studies indicated that the transcription of two-thirds of genes induced during zoosporogenesis relied on calcium signaling. Overall, we concluded that spore formation and germination involves the staged expression of a large subset of the transcriptome, commensurate with the importance of spores in the life cycle. Analyses revealed dynamic changes in genes involved in pathogenicity, metabolism, and signaling, with diversity in expression observed within members of multigene families and between isolates. We also completed an improved genome assembly for Phytophthora infestans, mapped the location of genes determining fungicide resistance, and assessed the role of copy number variation in fungicide resistance.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Ah-Fong AM, Shrivastava J, Judelson HS. 2017. Lifestyle, gene gain and loss, and transcriptional remodeling cause divergence in the transcriptomes of Phytophthora infestans and Pythium ultimum during potato tuber colonization. BMC Genomics 201718:e764
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Judelson HS. 2017. Metabolic diversity and novelties in the oomycetes. Annual Review of Microbiology, 71:21-39.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Ah-Fong AM, Kim KS, Judelson HS. 2017. RNA-seq of life stages of the oomycete Phytophthora infestans reveals dynamic changes in metabolic, signal transduction, and pathogenesis genes and a major role for calcium signaling in development. BMC Genomics 18, e198.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Abrahamian M, Ah-Fong AMV, Davis C, Andreeva K, Judelson HS. 2016. Gene expression and silencing studies in Phytophthora infestans reveal infection-specific nutrient transporters and a role for the nitrate reductase pathway in plant pathogenesis. PLoS Pathogens 12, e1006097.
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