Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The target audience consisted mainly of students in higher education, such as undergraduate and graduate students, as well as university faculty of different academic ranks. Means of communication included publications in peer-reviewed journals and invited seminars in academic institutes. Changes/Problems:Some of the experiments that were planned fo this reporting period could not be performed because of the suspension of all non-critical on-campus research activities due to the COVID-19 pandemic. We have resumed part of our research activities but we are still ramping-up research. There are no other changes to the project. What opportunities for training and professional development has the project provided?A graduate student (Ching-Jung Lin) was hired on the project in September 2020. The student's project is on the functional characterization of the Ecp2 fungal core effector family in Cladosporium fulvum and other fungi. A second graduate student (Alex Zaccaron) with a background in bioinformatics has also been involved in this project. The student performed the assembly and annotation of the Pseudocercospora fuligena genome as well as the mining of this genome for effector proteins. Finally, an MSc student (Que van Huynh) has also been working on this project during this reporting period. The MSc student performed experiments related to the functional analysis of the Ep2 and Avr4 core effector families in C. fulvum. How have the results been disseminated to communities of interest?Results obtained in this HATCH project have been submitted for publication in the international peer-reviewed journal Science Advances. (Citation: "Chen LH, Kra?un SK, Nissen KS, Mravec J, Jørgensen B, Labavitch J, Stergiopoulos I. A diverse member of the fungal Avr4 effector family interferes with Ca2+-crosslinking in plant cell walls". Submitted). The manuscript is currently undergoing a second round of reviews. The manuscript was submitted in 2019 at Nature Communications but after two rounds of reviews, it was finally not accepted for publication in this journal. After revising the manuscript and adding more experimental data to it, part of which were generated during this report period, the manuscript was submitted for publication to Science Advances. A manuscript that describes the genome of Pseudocercospora fuligena has also been published in the international peer-reviewed journal Molecular Plant-Microbe Interactions (Citation: Zaccaron AZ, Stergiopoulos I. First Draft Genome Resource for the Tomato Black Leaf Mold Pathogen Pseudocercospora fuligena. Molecular Plant-Microbe Interactions. 2020 Dec 23;33(12):1441-5). The Research described in this HATCH project has also been communicated by means of an oral presentation at the students and faculty of the department of Plant Pathology, at the National Chung-Hsing University, in Taiwan in November 19 2020. A seminar on general principles of plant immunity was also given on the same date at this institution. Both the seminars were via zoom. Finally, our effectors work was presented at the department of Plant Pathology at UC Davis, during the departmental annual retreat in September 22. What do you plan to do during the next reporting period to accomplish the goals?The focus for the next reporting period will remain on the functional analysis of the Ecp2 effector family. The wok was initiated during the previous reporting period but some of the experiments need to be repeated or had to be placed on hold due to COVID-19 pandemic related suspension of all non-critical research activities on campus. Specifically, we will continue our efforts to generate knock-out mutants of the CfEcp2 and CfEcp2-2 paralogs present in Cladosporium fulvum as well as Pseudocercospora fuligen (PfEcp2-3 only). New gene knock-out constructs for the targeted replacement via homologous recombination of the three Ecp2 paralogs by the hygromycin resistance cassette are currently being prepared by the graduate student working on this project, whereas attempts to generate deletion mutants of the CfEcp2 and CfEcp2-2 paralogs using our previous constructs are being continued. In addition, constructs for the transient expression by agroinfiltration and PVX agroinfection in various Nicotiana spp. species and tomato of the three Ecp2 paralogs are being prepared. These will be used to screen wild tomato accessions an Nicotiana spp. for the presence of cognate resistance genes against. Wild tomato accessions will be obtained from the Tomato Genetics Resource Center (TGRC) at UC Davis whereas various Nicotiana species have been obtained from the US Nicotiana Germplasm Collection.
Impacts
What was accomplished under these goals?
We have previously reported on the functional and structural characterization of the Avr4 core effector family, whose founding member was cloned from the tomato pathogen Cladosporium fulvum (CfAvr4). CfAvr4 is a secreted fungal effector protein that utilizes a carbohydrate-binding module of family 14 (CBM14) to bind to chitin that is present in fungal cell walls and protect it from host chitinases during infections. Functional orthologues of CfAvr4 are abundantly present in a number of fungal species that are pathogens on various plants hosts. Moreover, a gene duplication within the Avr4 core effector family generated two effector lineages (Avr4 and Avr4-2) with distinct carbohydrate-binding specificities, localization patterns, and biological functions during pathogenesis. We have shown, for example, that PfAvr4-2, a paralog of PfAvr4 in the tomato pathogen Pseudocercospora fuligena, contains a CBM14 domain but it does not bind chitin or protect fungi against chitinases. Instead, PfAvr4-2 interacts with highly de-esterified pectin in the plant's middle lamellae or primary cell walls and interferes with Ca2+-crosslinking of homogalacturonan (HG) chains at cell-cell junction zones. This, in turn, synergizes the activity of fungal endo-polygalacturonases and further aids mycelial growth in the apoplastic space by loosening the cell wall structure and reducing cell-cell adhesion. During this reporting period we continued the functional characterization of PfAvr4-2 and performed a biochemical analysis of the two PfAvr4-2-expressing tomato lines that we had previously generated. Specifically, in collaboration with Prof. B. Jørgensen at the department of plant and environmental sciences, at the University of Copenhagen, Frederiksberg, in Denmark we performed a Comprehensive Microarray Polymer Profiling (CoMPP) analysis and monosaccharide composition analysis of the cell walls of the two PfAvr4-2-expressing tomato lines and of the control moneymaker lines, in order to compare and contrast their biochemical composition. The results showed that changes in the HG crosslinking of the plant cell walls in the PfAvr4-2 transgenic lines are to some extend compensated by altered production and higher intermolecular crosslinking of some non-HG cell wall components, such as xylan, arabinoxylan and possibly proteoglycans that are capable of forming cross-links in the plant cell wall. However, these changes are relatively minor, suggesting that, apart from the disruption of Ca2+-crosslinking of homogalacturonan (HG) chains at cell-cell junction zones, there are no major alterations in the overall composition of the plant cell walls of the PfAvr4-2 transgenic lines. Next to performing a biochemical analysis of plant cells, several attempts to obtain a crystal structure of PfAvr4-2 were also made but these were unsuccessful so far. We have thus computationally modelled the structure of PfAvr4-2 on the tertiary structures of PfAvr4 and CfAvr4 and identified discrepancies that could explain the differences in substrate specificity. These results will be used as a basis for subsequent mutational analysis of PfAvr4-2. A genome assembly and annotation of Pseudocercospora fuligena have also been produced. No previous representative genome was available for P. fuligena, which makes this the first draft genome for this species. The P. fuligena genome produced contains 348 contigs and 13,764 predicted protein-coding genes with an estimated completeness of 98%. Among the predicted genes there were 179 candidate effectors, including the two paralogs of Avr4 (PfAvr4 and PfAvr4-2) and the three paralogs of Ecp2 (PfEcp2, PfEcp2-2 and PfEcp2-3). However, two of the three Ecp2 paralogs present in P. fuligena are shown to be pseudogenes. Current efforts are focused on functionally characterizing the Ecp2 core effector family from fungi, whose members are broadly distributed in plant, animal, and human pathogens. We have previously reported the presence of three Ecp2 paralogs in C. fulvum (i.e. CfEcp2, CfEcp2-2, CfEcp2-3). Attempts to generate deletion mutants of these paralogs in C. fulvum and to further characterize their virulence on tomato plants were initiated during the previous reporting period and were continued in this one. To this end, constructs for the targeted replacement via homologous recombination of the three Ecp2 paralogs by the hygromycin resistance cassette have been produced and a deletion mutant of CfEcp2-3 was successfully generated. Analysis of the gene deletion mutant with respect to its virulence on tomato plants has shown that CfEcp2-3 is a likely virulence factor. Attempts to generate knock-out mutants of CfEcp2 and CfEcp2-2 have been made as well but these were unsuccessful as only ectopic transformants were been obtained. The experiments are repeated by generating new constructs that differ in the length of flanking regions for homologous recombination of the three Ecp2 paralogs by the hygromycin resistance cassette.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Chen LH, Kra?un SK, Nissen KS, Mravec J, J�rgensen B, Labavitch J, Stergiopoulos I. A diverse member of the fungal Avr4 effector family interferes with Ca2+-crosslinking in plant cell walls.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zaccaron AZ, Stergiopoulos I. First Draft Genome Resource for the Tomato Black Leaf Mold Pathogen Pseudocercospora fuligena. Molecular Plant-Microbe Interactions. 2020 Dec 23;33(12):1441-5.
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The results on the functional characterization of the PfAvr4-2 effector protein that are presented in this report have been mainly communicated to the general scientific community through international conferences and invited talks at academic institutions. In specific, they were presented as oral talks in at least three occasions: 1. Invited Speaker. "Biochemical, structural, and functional insights into fungal core effector proteins". The Sainsbury Laboratory (TSL), UK, October 11 2019 2. Oral presentation: "Biochemical, structural, and functional insights into fungal core effector proteins". Bay Area Meetup for Tomato Plant Pathology (BAMTOPP), Berkeley, CA, USA, May 29th 2019 3. Invited Talk. Emergence of a novel effector function through gene duplication and functional diversification in the fungal Avr4 core effector family. 30th Fungal Genetics Conference, Pacific Grove, CA, USA, March 12-17 2019 Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two post-doctoral fellows (i.e. Dr Li-Hung Chen and Dr. Jonathan Nino Sanchez) and one MSc student (Que van Huynh) were engaged in the projects described in this report. Specifically, Dr Chen has worked on the functional characterization of the PfAvr4-2 effector, whereas Dr Sanchez has worked on the functional analysis of the Ep2 effector family in C. fulvum in collaboration with the Master's student. How have the results been disseminated to communities of interest?Results described in this HATCH project have been submitted for publication and are currently under peer review in Nature Communications. They have also been broadly communicated to the general scientific community through conferences or invited talks, as indicated under "target audience". What do you plan to do during the next reporting period to accomplish the goals?Our focus during the next reporting period will be towards analyzing the function of the three Ecp2 paralogs present in C. fulvum. As aforementioned, gene knock-out constructs for all three genes have already been produced and currently fungal transformations are underway for the homologous replacement of the three genes by the hygromycin resistance cassette. If loss-of-function mutants are obtained, these will be phenotypically characterized with respect to their infectivity on tomato plants. Constructs for the complementation of the loss-of-function mutants, once these are obtained, by the wild-type Ecp2 paralogs are also currently being constructed. Finally, although CfEcp2 is known to be recognized in tomato by the cognate Cf-Ecp2 resistance gene that originates from Solanum pimpinellifolium, cognate genes for CfEcp2-2 and CfEcp2-3 have not been identified yet. For this purpose we have generated constructs for the heterologous expression of these two genes in tomato and these will be used to screen wild tomato accessions for the presence of cognate resistance genes against them. Wild tomato accessions will be obtained from the Tomato Genetics Resource Center (TGRC) at UC Davis.
Impacts
What was accomplished under these goals?
Effectors are small-secreted proteins that promote virulence during pathogen infections. Although key to microbial infections, the intrinsic function of effectors remains for most a mystery, as they lack sequence annotations that could provide hints to their intrinsic function and localization during pathogenesis. To date, only a handful of secreted into the apoplast fungal effectors have been functionally elucidated from extracellular pathogens and all are shown to fulfil their role in pathogenesis outside the host cells, by targeting plant- or self-derived components. A prominent example is the CfAvr4 effector protein from the tomato pathogen Cladosporium fulvum, which utilizes a carbohydrate-binding module of family 14 (CBM14) to bind to chitin that is present in fungal cell walls and protect it from host chitinases during infections. Unlike the vast majority of effectors that are species-specific, homologs of CfAvr4 are found in a plethora of other fungal species with diverse host-specificities and nutritional life-styles. We have previously reported on the functional and structural characterization, either alone or in complex with chitin, of two Avr4 family members from the tomato pathogens Pseudocercospora fuligena (PfAvr4) and C. fulvum (CfAvr4). We have also shown that gene duplication in the Avr4 effector family produced two effector lineages with distinct carbohydrate-binding specificities, localization patterns, and biological functions during pathogenesis. Specifically, we had functionally characterized PfAvr4-2, a paralog of PfAvr4 in P. fuligena, and showed that although it contains a CBM14 domain, it does not bind chitin or protect fungi against chitinases. Instead, PfAvr4-2 interacts with highly de-esterified pectin in the plant's middle lamellae or primary cell walls and interferes with Ca2+-crosslinking of homogalacturonan (HG) chains at cell-cell junction zones. This, in turn, synergizes the activity of fungal endo-polygalacturonases and further aids mycelial growth in the apoplastic space by loosening the cell wall structure and reducing cell-cell adhesion. The above described analysis of the function of PfAvr4-2 was based on cytological observations using a series of labelling protocols and interfering substances that provided a strong correlation between the localization of PfAvr4-2 at de-esterified HG locations, and its possible role in the prevention of cell wall fortifications by Ca2+-crosslinking. In this HATCH project, we provided strong evidence that the our results are physiologically relevant during infections by generating transgenic tomato plants overexpressing PfAvr4-2 and further analyzing their mechanical, developmental, cytological, and biochemical properties. Specifically, biochemical analysis of two PfAvr4-2 overexpressing lines showed that they contain fewer so-called 'egg-boxes', as a result of reduced Ca2+-crosslinking of de-esterified pectin in the plant's middle lamellae. Egg-boxes are crucial to plant development as they control cell-cell adhesion, porosity, stretching and elasticity. Indeed, analysis of the mechanical properties of the PfAvr4-2 overexpressing lines showed that they are less stiff and more elastic that the wild-type plants. As a result the plants grow taller and have wider leaves but they are also considerably more frail and susceptible to pathogen attacks. Further cytological observations also revealed that transgenic plants suffer a less defined cell-shape, have more plastic cell walls, and reduced cell-cell adhesion. Overall, the analysis of the detailed phenotypic and biochemical characterization of the PfAvr4-2 transgenic lines, corroborated our previous cytological observations and provided the missing evidence for the physiological relevance of PfAvr4-2 during pathogen infections. In this reporting period we have also initiated work on the Ecp2 core effector family from C. fulvum. We have previously shown that homologs of the Ecp2 effector protein from C. fulvum are members of an ancient and widely distributed within the fungal kingdom superfamily of effectors. Three also paralogs of Ecp2 are present in the genome of C. fulvum and other Dothideomyceres, which are designated as CfEcp2, CfEcp2-2, and CfEcp2-3. The intrinsic function of these paralogs as well as their contribution to virulence remains unknown, but given the wide distribution across fungi it is expected that they likely facilitate infections across a number of hosts. To test this hypothesis, loss-of-function of mutants of the three Ecp2 paralogs will be generated in C. fulvum and their virulence will be examined on susceptible tomato plants. Gene-knock-out constructs have been produced for all three gene and fungal transformations are currently under way. If gene knock out mutants are obtained these will be next tested for virulence on susceptible tomato plans.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Functional elucidation of a novel effector that pertrubs plant cell walls during fungal infections
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Progress 10/11/17 to 09/30/18
Outputs
Target Audience:The results form this HATCH project have been mainly communicated to the general scientific community through international conferences and invited talks at academic institutions. In specific, they were presented as oral talks in at least two occasions: 1) Invited Speaker. "Evolutionary, structural, and functional insights into fungal core effector proteins". Linnean Centre for Plant Biology in Uppsala, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden. September 13 2018 2) Invited by the Chair Talk. "Biochemical, structural, and functional insights into fungal core effector proteins". Gordon Research Conference (GRC) on Cellular and Molecular Fungal Biology "Harnessing Diversity to Understand Molecular Mechanisms, Pathogenicity, and Fungal Factories', June 17-22, Holderness, NH, USA. June 21, 2018 Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate and one mater's student have been mainly involved in this work. The graduate student submitted his PhD dissertation at the end of December 2018 and the master's student started his work on effectors in October 2018. How have the results been disseminated to communities of interest?The results described in this HATCH project have been published or are currently submitted in prestigious international peer-reviewed journal such as PloS Pathogens, and they have also been broadly communicated to the general scientific community through conferences or invited talks, including as an invited talk during a Gordon Research Conference (GRC). What do you plan to do during the next reporting period to accomplish the goals?We plan to continue investigating the biology of core effector proteins by shifting our attention to the fungal Ecp2 family of core effectors. Specifically, we have previously discovered that homologs of the Ecp2 effector protein from the tomato pathogen C. fulvum are members of an ancient and widely distributed within the fungal kingdom superfamily of effectors. The driving force behind the patchy distribution of Ecp2 homologs in different fungal species is still unknown but likely relates to their uncharacterized intrinsic function. One possibility is that Ecp2 genes are gained or lost after speciation events according to niche-specific selection pressures. Although the Cf-Ecp2 resistance gene from tomato has not be cloned yet, heterologous expression of Ecp2 effector proteins from different fungi in a Cf-Ecp2 tomato line induced a strong HR, indicating that they are likely to be perceived by the putative Cf-Ecp2 immune receptor. Same as Cf-4, our findings suggest that Cf-Ecp2 could be functioning as a PRR that recognizes discrete Ecp2 homologs from different fungi.
Impacts
What was accomplished under these goals?
Effectors are microbial-derived secreted proteins with an essential function in modulating host immunity. Most effectors were until recently assumed to be species-specific that originated late in a pathogen's evolutionary history to facilitate disease on specific hosts. However, research in our lab and elsewhere has revealed the presence of abundant homologous "core" effector proteins across fungi, thus challenging the "species-specific" paradigm in plant-pathogen interactions. Further on, we have shown that despite their low sequence similarity core effectors can be collectively perceived by a single cognate immune receptor, thus generating novel opportunities for disease control. In this HATCH project, we sought to provide answers to critical questions regarding the biology of core effector proteins, including whether their biological function and contribution to virulence are conserved among different fungal species, and what is the molecular basis for their broad-recognition by cognate plant immune receptors. To answer these questions we have structurally, biochemically, and functionally analyzed three members of the fungal Avr4 effector family, whose founding member (i.e. CfAvr4 from the tomato pathogen Cladosporium fulvum) was shown to promote parasitism thorough binding chitin in fungal cell-walls and protecting it from chitinases. Members of this family are shown to be broadly distributed in fungi with different life-styles and, remarkably, despite their low sequence identity, many are recognized by the cognate Cf-4 resistance protein from tomato. To this date, we have successfully solved in collaboration with Dr Andrew Fisher from the Chemistry Department, the crystal structure of two Avr4 family members either alone (i.e. PfAvr4 the tomato pathogen Pseudocercospora fuligena) or in complex with chitin (i.e. CfAvr4) and by subsequent structure-guided site-directed mutagenesis we have provided evidence for the structural separation between the virulence and avirulence properties in this effector family. Further on, we have found that gene duplication has been a major driver of evolution within this effector family that generated paralogs with a novel function that, to our knowledge, has not been described before. Specifically, we have functionally characterized PfAvr4-2, a paralog of PfAvr4 in P. fuligena, and have shown that although it contains a CBM14 domain, it does not bind chitin or protect fungi against chitinases. Instead, PfAvr4-2 interacts with highly de-esterified pectin in the plant's middle lamellae or primary cell walls and interferes with Ca2+-crosslinking at cell-cell junction zones, thus loosening the plant cell wall structure and further synergizing the activity of pathogen secreted endo-polygalacturonases. To the best of our knowledge, this is the first example of a microbial effector that perturbs plant cell walls in order to assist infections.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Functional elucidation of a novel effector that perturbs plant cell walls during fungal infections.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Structure of the Cladosporium fulvum Avr4 effector in complex with chitin uncouples the ligand-binding function from Cf-4 recognition
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Biochemical, structural, and functional characterization of the Avr4 core effector family in fungi
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