Source: PENNSYLVANIA STATE UNIVERSITY submitted to
GENOMIC APPROACHES TO THE IDENTIFICATION OF FUNGAL PLANT PATHOGENS AND UNDERSTANDING HOST DEFENSE RESPONSE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1000117
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jul 1, 2013
Project End Date
Jun 30, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802
Performing Department
Plant Pathology & Environmental Microbiology
Non Technical Summary
Fusarium, Phytophthora and Magnaporthe are three of the most important genera of plant disease-causing microorganisms. Confounding solutions to the problems they case, which range from Fusarium Head Blight of wheat and barley to late blight of potato, is our lack of knowledge about their global diversity, and a lack of available tools that catalog and organize what we do know about it. This project seeks to provide 1) a DNA-based classification and identification system for Fusarium and Phytophthora diversity using molecular phylogenetics and 2) data and basic information about the fungi via the internet that is useful for frontline researchers dealing with problems caused by these organisms. In addition, we focus on the molecular basis of one of the world's most important plant diseases, rice blast caused by Magnaporthe oryzae, to elucidate the basic aspects of how the fungus and plant interact, with a focus on how the plant responds and defends itself against the pathogen.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21240201102100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1102 - Mycology;
Goals / Objectives
Enhance current cyberinfrastructural resources for fungal identification (Phytophthora Database and Cyberinfrastructure in Fusarium) toInclude more sequence data and more isolates.Provide more accession data and useful information associated with isolates/identification results.Produce an updated molecular phylogeny of the genus FusariumUtilize additional, phylogenetically informative loci identified via comparative genomics.Better resolve the phylogenetic boundary of the genus.Better resolve phylogenetic relationships among Species Complexes within the genus.Facilitate the characterization of novel and emerging Fusarium and Phytophthora pathogens and toxin producers by robust molecular phylogeneticsPlace newly discovered species in a well-resolved phylogenyUse highly informative molecular markersTranslation elongation factor 1-alphaLargest and second largest RNA polymerase II B-subunit genes (RPB1 and RPB2)Additional loci, some to be discovered using comparative genomicsProduce taxonomic updates of Fusarium that will help frontline researchers in agriculture and other fieldsProvide an electronic and printed publication summarizing the Species Complexes in FusariumPropose and summarize an updated taxonomic summary of Fusarium available in printed and electronic formTo understand the mechanisms of pathogen virulence and host immunity during the rice-Magnaporthe interaction and to develop novel strategies for genetic improvement of rice disease resistance:Characterize rice proteins targeted by fungal effectorsBiochemical and functional analyses of stress responsive MAP kinases and creatine phosphokinases and their substratesGenetically modify rice for enhanced disease resistance and stress tolerance
Project Methods
Objectives 1-4: Molecular markers for phylogenetics are generally developed using available complete genome sequences, as illustrated in recent work (Blair et al. 2008). New genomic methods involve identification of orthologous genes from a set of complete genome sequences, and then designing primers that are ortholog-specific. Primers are designed corresponding to sites within genes that are conserved across the taxon of interest, usually located in exon regions that flank introns that are informative at the species level. Phylogenetic relationships are inferred using a consensus approach, utilizing maximum parsimony, Bayesian and Maximum Likelihood methods. Bootstrap as well as Bayesian posterior probabilities are used as indicators of node support. Intraspecific polymorphism identified in these markers can be used for population analyses, as well as nucleotide polymorphism identified in other loci (especially intergenic regions), and tandem repeats (i.e., microsatellites). Species boundaries are inferred using principles of genealogical concordance (Taylor et al. 2000). Resulting data, along with any available phenotypic traits of each species and individual isolates within species (such as morphology, growth characteristics, profiles of secondary metabolites) will be archived in appropriate databases in a format that can be easily searched and updated through the internet. These databases will support pathogen identification as outlined in Goal 3. While multiple loci are generally required to infer robust phylogenies and to recognize species boundaries, a single locus can be implemented as a useful identification tool. A ~700 bp portion of the 5' end of the translation elongation factor 1-alpha gene has proven to be a very powerful tool for identifying Fusarium isolates to phylogenetically defined species (Geiser et al. 2004). In recent years, we have added full DNA sequences of the largest and second largest B-subunits of the RNA Polymerase II genes, which despite lacking introns are highly informative even at the species level (O'Donnell et al., 2013). In the genus Phytophthora, the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene repeat have proven to be the best frontline tool for identification, but protein-coding genes such as HSP90 have potential for greater resolution with no loss of breadth of utility. Sequence based identification of unknown pathogens using these loci involves DNA sequencing of the target locus, followed by BLAST searches to identify best hits against a database of sequences corresponding to vouchered, publicly available specimens. Objective 5: (a) To characterize rice proteins targeted by fungal effectors - A number of rice proteins have been isolated by yeast two-hybrid screening and shown to interact with a family of Nep1-like proteins (MoNLPs) or a family of zinc fing proteins (ZF) by bimolecular fluorescence complementation (BiFC) or co-immunoprecipitation (co-IP) in vivo. Gain- and loss-of-functional analyses via reverse genetics will be carried out to determine the function of these host targets in signal transduction and rice disease resistance and susceptibility. In addition, the host target protein complexes are being characterized and will be functionally analyzed to determine their roles in perception of fungal effectors and initiation of host signaling cascades. (b) Biochemical and functional analyses of stress-responsive rice MPKs and CPKs and their substrates - Several stress-responsive rice MPKs and CPKs have been characterized and shown to play important roles in mediating host defense response. In addition, substrates of a MPK and a CPK have been isolated by solid-phase phosphorylation screening. Biochemical and functional characterization are being carried out with some of these substrates. More comprehensive genetic and functional analyses will be conducted to elucidate the mechanism of MPK- and/or CPK-mediated signaling crosstalks and their roles in rice biotic and abiotic stress tolerance. (c) to genetically modify rice crop for enhanced disease resistance and stress tolerance - Based on the new knowledge and tools, we plan to develop genetically modified rice with enhanced resistance to major rice diseases such as rice blast and sheath blights. Efforts will be made to avoid negatively affecting abiotic stress tolerance or crop yield and quality.

Progress 07/01/13 to 06/30/18

Outputs
Target Audience:Phytophthora Database and Fusarium-ID, built and curated by Geiser, Kang and their USDA/ARS collaborators, continue to support researchers, extension educators and disease diagnosticians around the world in identifying Phytophthora and Fusarium pathogens. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two Ph.D. students in Kang lab, Bongsoo Park and Venky Moktali, gained informatics skills and publications by curating and improving Fusarium and Phytophthora databases. Two other graduate students of Kang, Ningxiao Li and Tareq Islam, learned how to process leaf bait samples to diagnose P. ramorum. Jung-Eun Kim, a postdoctoral associate in Kang lab, experienced how molecular methods can be applied to diagnose and identify various pathogens. Former PhD students Dylan Short and Christopher Smyth were trained in Fusarium systematics and phylogenetics, resulting in the descriptions of new Fusarium species and identifying their presence in plumbing systems and sea turtle nests. Undergraduate students Clara Miller, Shane Pusey and Lucy Lagoze were trained in generating phylogenetic sequence datasets for Fusarium species, and Lucy Lagoze was trained in extracting and curating molecular markers from Fusarium genome sequences. Two former PhD students, Bastian Minkenberg and Yueying Chen, were trained for molecular plant pathology, biochemistry, genetics, genome editing and bioinformatics skills. Two other PhD students, Matthew Wheatley and Justin Shih, have also gained considerable experience and skills in molecular biology, rice pathology, genetics and CRISPR/Cas9 genome editing. How have the results been disseminated to communities of interest?Publication, reports and meeting presentations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Our project completed key taxonomic studies on some of the most important Fusarium species, recognizing some as new species, reassigning species status of some, and providing clarification of long-standing names by narrowing their use to well-defined species. This work supports goals 1-4, adding to our databases of Fusarium sequence diversity, provides updated phylogenies, facilitates characterization of novel pathogens, and updates taxonomy. 'F. solani' is a name that scientists have known to be applied to many species (a "species complex" we now call the F. solani Species Complex (FSSC)) for several decades, which necessitated the assignment of this name to one of those species, connected to its original description in 1842 by von Martius. We isolated Fusarium species from dry-rotted potatoes in the type locality and found it dominated by one phylogenetically recognizable species in FSSC, an unnamed species referred to as FSSC 5. This species fits von Martius' description well, and represents a commonly occurring, soil-inhabiting species that is also known to be a human pathogen, as does 'F. solani' as it is commonly used. We identified one isolate of F. solani from this study to be an epitype for the name, and delimited the species to FSSC 5, which provides a focus of comparison for further taxonomic work in delimiting additional taxa. Two such taxa are the most common two human pathogenic Fusarium species that occur in plumbing systems, previously referred to as F. solani, and phylogenetically recognized as FSSC 1 and FSSC 2. FSSC 1 was connected to F. solani var. petroliphilum and elevated to species status. FSSC 2 was connected to a taxon described as Cephalosporium keratoplasticum, which was recombined into Fusarium as F. keratoplasticum. The use of these names, as opposed to the catch-all 'F. solani,' has facilitated the discovery of connections to other species: F. keratoplasticum and F. petroliphilum are ecologically distinguished from a common soilborne FSSC species associated with human infections, F. falciforme (FSSC 3+4). F. keratoplasticum was known only from clinical infections and highly anthropogenically influenced substrates such as plumbing and indoor environments until 2013, when it was discovered in association with fouled sea turtle nests worldwide. Additional work from Geiser group has shown F. keratoplasticum and F. falciforme both to be dominant components of beach sand around sea turtle nests in Florida, suggesting that these species are a normal associate. Additional work using an expanded suite of molecular markers revealed that a worldwide clone complex within F. keratoplasticum dominates in plumbing in the US, as well as in human infections. Also toward goals 1-2, we assembled new genomics-based datasets that will facilitate 1) improved knowledge of diversity among the vascular wilt pathogen group Fusarium oxysporum, 2) discovery of differences among pathogenic and non-pathogenic strains of F. oxysporum, and 3) development of diagnostic tools useful for rapid identification of novel and previously known pathogens. 41 highly informative sequence-based markers were developed from publicly available datasets, which together provided a highly resolved phylogeny for this group. We are currently working on standard PCR/Sanger and next-generation methods for extracting the entire suite of marker sequences for strains, as well as subsets. We are collaborating with other scientists in the development of diagnostic tools that will be useful for fast and reliable identification of specific vascular wilt pathogens within F. oxysporum. Working with a PA Department of Agriculture (PDA) program that monitors diverse crops and environments in the state, we used sequence data to identify Phytophthora species associated with diseased plants. This work achieved aims applied to goals 1 and 3. Many of these were collected by PDA plant inspectors in the state diagnostic clinic. Ongoing work in the clinic produced cultures dating back to the 1980s, consisting of many Phytophthora cultures. An ongoing problem in the PA Christmas tree industry is Phytophthora root rot, particularly on true fir (Abies spp.), with heavy losses in seedling nurseries and plantations. Applying our molecular diagnostic tools to 225 cultures, we were able to infer historical (1988-2013) host-pathogen patterns, with Fraser fir, concolor fir and balsam fir being the most commonly infected hosts, andP. kelmania,P.cactorum,P.cinnamomi andP.sansomeanathe most commonly isolated Phytophthora species. P.chlamydospora,P.europaeaandP.colocasiaehave been isolated only once. This information will help guide seasonalPhytophthoraroot rot inspection in Pennsylvania, and it provides a long-term framework for monitoring the disease. In response to repeated shipments of P. ramorum-infected ornamental plants to nurseries around the nation, the USDA-Forest Service has been surveying forests in 20 states to check whether the pathogen escaped from nurseries to forest vegetation. Kang and collaborators have performed diagnosis of P. ramorum in samples collected from streams in 17 states using APHIS-approved PCR protocols. They also have cultured and purified Phytophthora and related genera from submitted samples, generating a library of >2,000 Phytophthora and some other oomycetes (e.g., Pythium, Phytopythium). Analysis of the collection would provide a crucial insight into the spatial and temporal diversity and ecology of Phytophthora species associated with diverse forests. Rice blast, caused by the fungus Magnaporthe oryzae, is perhaps the most serious disease on the most important food crop on earth, and thus a major threat to food security. Necrosis- and ethylene-inducing protein 1-like proteins (NLPs) represent a class of conserved microbial effectors with cytotoxic activities in dicot plants. They are also present in M. oryzae but their effects on monocots are poorly understood. Applied to goal 5, we demonstrated the role of MoNLPs in causing necrotic cell death in monocots and identified a rice ubiquitin-like protein that interacts with MoNLPs and mediates the cell death. We have also characterized a family of M. oryzae zinc-finger effectors and identified their interacting proteins in rice plants. One of the interacting proteins is part of the plant HIRA/AS1/AS2 silencing system that was shown to be involved in rice defense gene expression and host immunity. In addition, we have adapted and improved the CRISPR/Cas9 technology for genome editing in plant and fungal systems, which include the development of bioinformatic tools for predicting gRNA specificity, improvement of multiplex editing and functional elucidation of two MAP kinases involved in rice embryo and pollen development, as well as biotic and abiotic stress tolerance. CRISPR/Cas9 was also used to help validate a rice gene conferring broad-spectrum resistance to most US rice blast isolates in the US.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhao, H., Wang, X., Jia, Y., Minkenberg, B., Wheatley, M., Fan, J., Jia, M. H., Famoso, A., Edwards, J.D., Wamishe, Y., Valent, B., Wang, G.-L., Yang, Y. 2018. The rice blast resistance gene Ptr encodes a novel protein and confers broad spectrum disease resistance. Nature Communications 9:2039.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Li, N. and Kang, S. (2018) Do volatile compounds produced by Fusarium oxysporum and Verticillium dahliae affect stress tolerance in plants? Mycology 6: 166-175.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Kim, H., Kim, J., Son, H., Frailey, D., Cirino, R., Lee, Y.-W., Duncan, R., Czymmek, K., and Kang, S. (2018) Roles of three Fusarium graminearummembrane Ca2+ channels in the formation of Ca2+ signatures, growth, development, pathogenicity and mycotoxin production. Fungal Genetics & Biology 111: 30-46.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Phytophthora Database and Fusarium-ID, built and curated by Geiser, Kang and their USDA/ARS collaborators, continue to support researchers, extension educators and disease diagnosticians around the world in identifying Phytophthora and Fusarium pathogens. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students, Ningxiao Li and Tareq Islam, and one postdoc, Jung-Eun Kim, in Kang lab practiced how to detect Phytophthora ramorum in leaf bait samples collected from multiple states. Three graduate students, Yueying Chen, Matthew Wheatley and Justin Shih, have learned techniques for studying molecular plant-microbe interactions and trained to perform CRISPR/Cas9 editing for plant genome engineering. How have the results been disseminated to communities of interest?Publication, reports and meeting presentations. What do you plan to do during the next reporting period to accomplish the goals?Kang will characterize if and how fungal volatile compound-mediated interactions affect biocontrol efficacy of Fusarium wilt in tomato using Trichoderma-based biocontrol agents. Yang will develop new tools and strategies to improve CRISPR/Cas editing techniques and produce transgene-free rice cultivars with enhanced disease resistance and other important agronomic traits. Geiser will complete a phylogenomic analysis of Fusarium oxysporum.

Impacts
What was accomplished under these goals? We assembled new genomics-based datasets that will facilitate 1) improved knowledge of diversity among the vascular wilt pathogen group Fusarium oxysporum; 2) discovery of differences among pathogenic and non-pathogenic strains, and 3) development of diagnostic tools useful for rapid identification of novel and previously known pathogens. 41 highly informative sequence-based markers were developed from publicly available datasets, which together provide a highly resolved phylogeny for this group. We are currently working on standard PCR/Sanger and next-generation methods for extracting the entire suite of marker sequences for strains, as well as subsets. We are collaborating with other scientists in the development of diagnostic tools that will be useful for fast and reliable identification of specific vascular wilt pathogens within F. oxysporum. The PA Department of Agriculture (PDA) monitors a diverse array of specialty crops, nurseries and surrounding environments to detect diseases of regulatory concern through plant disease inspection and diagnostic service. Samples of diseased plants, mainly collected by PDA plant inspectors, are processed at the PDA Plant Diagnostic Laboratory (PDAPDL) for diagnosis, often resulting in pathogen cultures. Among several groups of pathogens isolated and preserved since as early as 1980s, the largest group corresponds to Phytophthora. True fir trees (Abies spp.) are common hosts of Phytophthora species. Christmas tree root rot associated with Phytophthora species has been accountable for heavy losses in seedling beds and plantations for many years in Pennsylvania. We identified Phytophthora species isolated from root rot samples of Abies spp. using their sequences to understand their historical pattern of occurrence. Among 225 samples of Abies spp. diagnosed as Phytophthora root rot from 1988 to 2013, A. fraseri, A. concolor and A. balsamea were most commonly infected hosts, and P. sp. kelmania, P. cactorum, P. cinnamomi and P. sansomeana were most commonly isolated species, while P. chlamydospora, P. europaea and P. colocasiae have been isolated only once. This information will help guide seasonal Phytophthora root rot inspection in Pennsylvania. We characterized a rice ubiquitin-like protein that interact with M. oryzae NLP proteins and demonstrated its potential role in mediating cellular ubiquitination process and host cell death. We also characterized the HIRA/AS1/AS2 silencing complex targeted by M. oryzae zinc finger effectors and demonstrated its potential role in rice defense gene expression and immunity. Using CRISPR-enabled multiplex genome editing approach, we identified two MAP kinases essential for rice pollen and embryo development and analyzed additional two MAP kinases involved in rice biotic and abiotic stress tolerance. In addition, we performed targeted mutagenesis in a resistant rice cultivar using CRISPR/Cas9 and validated a rice blast resistance gene that confers broad spectrum resistance to most of M. oryzae isolates in the US.

Publications

  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Minkenberg, B., Xie, K., and Yang, Y. 2017. Discovery of rice essential genes by characterizing a CRISPR-edited mutation of closely related rice MAP kinase genes. Plant J. 89:636-648.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Minkenberg, B., Wheatley, M., Yang, Y. 2017. CRISPR/Cas9-enabled multiplex genome editing and its application. In Donald P. Weeks and Bing Yang, editors: Gene Editing in Plants, Vol 149, PMBTS, UK: Academic Press, pp. 111-132.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Hibbett, D., Abarenkov, K., K�ljalg, U., �pik, M., Chai, B., Cole, J., Wang, Q., Crous, P., Robert, V., Helgason, T., Herr, J.R., Kirk, P., Lueschow, S., ODonnell, K., Nilsson, R.H., Oono, R., Schoch, C., Smyth, C., Walker, D.M., Porras-Alfaro, A., Taylor, J.W., Geiser, D.M. 2016. Sequence-based classification of fungi. Mycologia 108: 1049-1068.
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Kang, S., Mansfield, M., Park, B., and Martin, F. (2016) Molecular identification of Phytophthora isolates using a DNA sequence based approach and the Phytophthora Database. In: K. Ivors (ed.) Laboratory Protocols for Phytophthora Species. APS Press, St. Paul, MN.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Helliwell, E.E., Wang, Q., and Yang, Y. 2016. Ethylene biosynthesis and signaling is required for rice immune response and basal resistance against Magnaporthe oryzae infection. Mol. Plant-Microbe Interact. 29: 831-843.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Phytophthora Database and Fusarium-ID, built and curated by Geiser, Kang and their USDA/ARS collaborators, continue to support researchers, extension educators and disease diagnosticians around the world in identifying Phytophthora and Fusarium pathogens. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students, Ningxiao Li and Vasileios Bitas, and two postdocs, Jung-Eun Kim and Hyeseon Kim, in Kang lab learned new techniques while performing the work on how F. oxysporum volatile compounds affect plant growth. How have the results been disseminated to communities of interest?Publication. What do you plan to do during the next reporting period to accomplish the goals?Kang will characterize if and how fungal volatile compounds affect biocontrol of plant diseases using Trichoderma species.

Impacts
What was accomplished under these goals? The epitypification of Fusarium solani was necessary to advance the systematics of an agriculturally important group of fungi. This name is applied to a complex of over 60 phylogenetically recognizable species (the Fusarium solani Species Complex, or FSSC), including many important pathogens of diverse, agriculturally important plants, but also to endophytes, human pathogens, soil saprophytes, and insect mutualists. Over half of the phylogenetic species remain unnamed, including some key plant pathogens. An important step in breaking open this taxonomic logjam is to properly recognize one of these species as the true Fusarium solani, by epitypifying the name in connection to a phylogenetic species that fits the original description. Based on new collections of FSSC isolates from its type locality (Central Europe) and host (causing dry rot of potato), we found that members of one specific phylogenetic species, known as FSSC 5, fit the original morphological description, and was capable of causing potato dry rot. FSSC 5 is also known as a cosmopolitan soil saprophyte, and occasional human pathogen. Phylogenetic analyses showed FSSC 5 to be a diverse, phylogenetically cohesive species, but also provided evidence for possible introgression with other members of FSSC. Because FSSC 5 fits the broadly applied concept of F. solani both morphologically and ecologically, it was determined that it is the best candidate for epitypification for this name. This epitypification opens the door for providing new names for the remaining unnamed members of the FSSC. We studied the role of fungal volatile organic compounds (VOCs) in plant growth using Fusarium oxysporum, a fungal species complex that often resides in the rhizosphere of assorted plants. Phylogenetically and ecologically diverse strains of F. oxysporum produce VOCs that augment shoot and root growth of Arabidopsis thaliana and tobacco. Growth responses of A. thaliana hormone signaling mutants and expression patterns of a GUS reporter gene under the auxin-responsive DR5 promoter supported the involvement of auxin signaling in F. oxysporum VOC-mediated growth enhancement. In addition, 1-naphthylthalamic acid, an inhibitor of auxin efflux, negated F. oxysporum volatile-mediated growth enhancement in both plants. Comparison of the profiles of volatile compounds produced by F. oxysporum strains that differentially affected plant growth suggests that the relative compositions of both growth inhibitory and stimulatory compounds may determine the degree of plant growth enhancement. Volatile-mediated signaling between fungi and plants may represent a potentially conserved, yet mostly overlooked, mechanism underpinning plant-fungus interactions and fungal niche adaption.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: ODonnell, K., Ward, T., Robert, V. A. R. G., Crous, P. W., Geiser, D. M., and Kang, S. (2015) DNA sequence-based identification of Fusarium: Current status and future directions. Phytoparasitica 43:583-595.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Li, N., Alfiky, A., Vaughan, M. M., Kang, S. (2016) Stop and smell the fungi: Fungal volatile metabolites are overlooked signals involved in fungal interaction with plants. Fungal Biology Reviews 30: 134-144.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Bitas, V., McCartney, N., Li, N., Demers, J., Kim, J.-E., Kim, H., Brown, K. M., and Kang, S. (2015) Fusarium oxysporum volatiles enhance plant growth by affecting auxin transport and signaling. Frontiers in Microbiology 6: 1248.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Inderbitzin, P., Putman, A. I., Subbarao, K. Kang, S. and Park, B. (2015) VertShield  an online resource for Verticillium species research. Phytopathology News (December) 49:160.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Provide a description of the target audience(s) reached by your efforts during this reporting period only. The target audience(s) you describe should include only those that your efforts reached during the current reporting period; this may mean that the audiences you list are only a subset of the all those you included on your project initiation. If you have no Target Audience to report, please indicate "Nothing to Report" below. We have generated data and tools that support pathogen identification and diagnosis, new species discovery, and molecular and cellular analyses of the mechanism underpinning pathogenicity and plant disease resistance. A collection of fungal plant pathogen databases built and curated by Geiser, Kang and their USDA/ARS collaborators have assisted researchers and extension educators in land grant institutions as well as researchers and regulators in state and federal agencies, such as Pennsylvania Department of Agriculture, USDA/APHIS and USDA/FS, in diagnosis of important diseases such as sudden oak death and implementing appropriate regulatory measures. The Fusarium and Phytophthora databases have been widely used by many disease diagnosticians in the U.S. and around the world. On average, each of these databases have been accessed ~1,300 times per month during this period. A newly developed database for the fungal genus Verticillium will further expand the target audience of this project. The study on the mechanism of plant-pathogen interactions and genome editing technology has facilitated plant pathologists, breeders and other researchers to improve agronomic traits such as crop disease resistance and stress tolerance. Our CRISPR-Plant website for designing guide RNAs has been accessed by over 6400 users from 90 countries and ~250 requests for CRISPR/Cas9 plasmid vectors were processed to facilitate genome editing research in many laboratories in the US and the world. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A former graduate student in Kang lab, Bongsoo Park, led the construction of VertShiled and helped curate these pathogen databases. Jung-Eun Kim, a postdoctoral associate in Kang lab, and Hye-Seon Kim, a former student and postdoctoral associate in Kang lab, led the analysis of calcium signatures in Fusarium oxysporum. The F. keratoplasticum population genetics work was carried out as part of the dissertation research of Dylan P.G. Short, who was supported by NIFA grant 2010-65110-20488. The project also provided training and research opportunity for four PhD students (Yueying Chen, Bastian Minkenberg, Kayla Echols and Matthew Wheatly), two postdoctoral associates (Kabin Xie and Xiangling Shen), and one senior research technologist (Qin Wang) in Yang lab. How have the results been disseminated to communities of interest?The results from this project have been disseminated to the scientific communities through publications, meeting presentations and websites. What do you plan to do during the next reporting period to accomplish the goals?Geiser have several ongoing projects characterizing agriculturally important Fusarium species that will move forward in the coming year. He will also characterize the identity and diversity of Phytophthora and Pythium species cultured from streams around 13 eastern states. Kang will improve the security of the three databases and expand the study on fungal calcium signaling by characterizing other species. Yang will seek to understand the molecular mechanism of rice immunity and disease resistance. He will continue to improve the CRISPR-Cas9 mediate genome editing tools and perform precision breeding to improve agronomic traits of agricultural crops such as rice, potato and mushroom.

Impacts
What was accomplished under these goals? F. keratoplasticum is a Fusarium species known exclusively from highly anthropogenically influenced environments, mostly from plumbing systems and from human infections. A worldwide sample of Fusarium keratoplasticum was analyzed using multilocus sequence-typing and population genetic analyses to infer patterns of recombination, clonality and interspecific hybridization. Results revealed evidence for an expanding clone complex, perhaps associated with adaptation to the human environment and its spread. Evidence was also identified for hybridization events between F. keratoplasticum and a related species also known from plumbing sources. These results have important implications for the movement and spread of these fungi in plumbing systems. VertShield incorporates several identification tools adapted from published studies and contain practical advice on how to identify and study Verticillium species. The tools include a morphological key, simplex and multiplex PCR assays, and a blast option for queries against a curated database of phylogenetically informative sequences. The General Identification Guide located under the Identification Tools & Guide tab informs users about how to generate and interpret data for species identification. A combination of these tools permits a robust evaluation of species affiliation, and for V. longisporum, lineage affiliation. VertShield also archives commonly used lab protocols (e.g., storage of Verticillium strains, pathogenicity tests) and images of typical Verticillium disease symptoms in various hosts. We welcome community contributions and suggestions, and envision that this effort will support Verticillium research, disease management and new species discovery. Spatial and temporal changes of cytoplasmic calcium ions ([Ca2+]c), caused by external stimuli, are known as the Ca2+ signature and presumably control cellular and developmental responses and pathogenicity. Multiple types of ion channels, pumps, and transporters on plasma and organellar membranes modulate influx and efflux of Ca2+ to and from the extracellular environment and internal Ca2+ stores to form Ca2+ signatures. Expression of a fluorescent protein-based Ca2+ probe, Cameleon YC3.60, in Fusarium oxysporum enabled Kang and collaborators to study how disruption of three Ca2+ channel genes, including FoCCH1, FoMID1 and FoYVC1, affects Ca2+ signature formation at polarized hyphal tips and whether specific changes in the Ca2+ signature caused by these mutations are related to a growth-related phenotype. Resulting mutants displayed altered amplitude, interval, and duration of Ca2+ pulses under various external Ca2+ concentrations as well as reduced sporulation and growth. Loss of FoMID1 and FoCCH1, genes encoding putative plasma membrane channel proteins, had major impact on Ca2+ signatures and growth, while disruption of FoYVC1, which encodes a vacuolar channel, only subtly affected both traits. Results from this study provide new insights into the underpinning of Ca2+ signaling in fungi and its role in controlling growth and also raise several new questions. Molecular, biochemical and genetic analyses have performed to characterize four rice proteins targeted by Magnaporthe oryzae Nep1-like protein and zinc finger effectors. Transgenic study and gene expression profiling have shown that these host proteins are involved in protein degradation and chromatin reconfiguration and play an important role in rice resistance to the blast pathogen. The study of downstream defense signaling events led to the identification of a novel phosphorylation pathway that connects calcium signaling to the MAP kinase activation. In addition, the polycistronic gRNA-tRNA strategy was developed to significantly enhance the CRISPR-Cas9 mediated genome editing capability and efficiency. To translation basic knowledge into agricultural productivity, precise genome engineering was performed with CRISPR-Cas9 to improve the agronomic traits of rice, potato and mushroom.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Xie, K., Chen, J., Wang, Q., and Yang, Y. 2014. Direct phosphorylation and activation of a mitogen-activated protein kinase by a calcium-dependent protein kinase in rice. Plant Cell 26: 3077-3089.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Boundy-Mills, K., Hess, M., Bennett, A. R., Ryan, M., Kang, S., Nobles, D., Eisen, J. A., Inderbitzin, P., Sitepu, I. R., Torok, T., Brown, D. R., Cho, J., Wertz, J. E., Mukherjee, S., Cady, S. L., and McCluskey, K. 2015. The United States Culture Collection Network (USCCN): Enhancing Microbial Genomics Research through Living Microbe Culture Collections. Applied & Environmental Microbiology 81: 5671-5674.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kim, H., Kim, J., Frailey, D., Nohe, A., Duncan, R., Czymmek, K., and Kang, S. 2015. Roles of three Fusarium oxysporum calcium ion (Ca2+) channels in generating Ca2+ signatures and controlling growth. Fungal Genetics & Biology 82: 145-157.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Xie, K., Minkenberg, B. and Yang, Y. 2015. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA processing system. Proc. Natl. Acad. Sci. USA 112: 3570-3575.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Through this project, we are collaborating with USDA/ARS laboratories and international institutions to study the phylogenetics of fungal pathogens and molecular mechanisms underpinning pathogenicity and plant disease resistance and to preserve resulting research materials, data and protocols in a format that can be easily shared and updated. A collection of fungal plant pathogen databases built and curated by Geiser, Kang and their collaborators have assisted researchers and extension educators in land grant institutions as well as researchers and regulators in state and federal agencies, such as Pennsylvania Department of Agriculture, USDA/APHIS and USDA/FS, in diagnosis of important diseases such as sudden oak death and implementing appropriate regulatory measures based on quick and accurate assessment of the risk of newly discovered pathogens. The Fusarium and Phytophthora collections and associated data also have been widely used by many disease diagnosticians in the U.S. and around the world. On average, Phytophthora Database and Fusarium-ID have been accessed ~1,500 times each per month during this period. The study on pathogen virulence, host resistance and translational biotechnology has helped plant pathologists and researchers who are interested in improving strategies for controlling crop diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The F. keratoplasticum population genetics work was carried out as part of the dissertation research of Dylan P.G. Short in Geiser lab. Two graduate students in Kang lab, Bongsoo Park and Venky Moktali, contributed to curating and improving pathogen databases and graduated in 2013. Jung-Eun Kim, a postdoctoral associate in Kang lab, learned about Phytophthora identification based on their gene sequences. The project in Yan lab provided excellent training opportunity for two PhD students (Yueying Chen and Bastian Minkenberg), one visiting graduate student (Xu Zhao), two postdoctoral associates (Kabin Xie and Xiangling Shen), and one senior research technologist (Qin Wang). How have the results been disseminated to communities of interest? The research results have been disseminated to the scientific communities through publications, meeting presentations and websites. What do you plan to do during the next reporting period to accomplish the goals? Geiser has several ongoing projects characterizing agriculturally important Fusarium species. Kang will continue to characterize the identity and diversity of Phytophthora species cultured from stream baits around eastern states, including Pennsylvania, and will also publically release a database for Verticillium species, a main group of soilborne fungal pathogens that threaten many specialty crop species. Yang plans to further characterize host target proteins involved in rice defense signaling and disease resistance and to elucidate the complex cross-talks between different signaling pathways. Yang will also improve multiplex genome editing and homologous recombination efficiency for site-specific integration and gene replacement in rice.

Impacts
What was accomplished under these goals? Fusarium keratoplasticum is a species known exclusively from highly anthropogenically influenced environments, mostly from plumbing systems and from human infections. A worldwide sample of F. keratoplasticum was analyzed using multilocus sequence-typing and population genetic analyses by Geiser and his collaborators to infer patterns of recombination, clonality and interspecific hybridization. Results revealed evidence for an expanding clone complex, perhaps associated with adaptation to the human environment and its spread. Evidence was also identified for hybridization events between F. keratoplasticum and a related species also known from plumbing sources. These results have important implications for the movement and spread of these fungi in plumbing systems. Kang and his collaborators have performed diagnosis of Phytophthora ramorum in leaf bait samples collected from streams in multiple eastern states using APHIS-approved PCR protocols. Since the beginning of 2014, 390 samples from 9 states have been received and processed. The number of samples from individual states ranged from 8 (TX) to 131 (GA). P. ramorum positive samples were collected in AL (3), FL (1), MS (2), and NC (1). In addition, Phytophthora was cultured from leaf bait samples, which will be identified via sequencing of their genes. They also characterized Phytophthora species associated with Christmas tree root rot in Pennsylvania, which is accountable for heavy losses in seedling beds and plantations for many years in PA. Plant inspectors of PA Department of Agriculture (PDA) have submitted symptomatic plants with the field information to the PDA Plant Disease Diagnostic Laboratory. Phytophthora isolates cultured from these samples have been deposited in the PDA culture collection since 1986 to present. Species identity of these Phytophthora isolates, their historical distribution and impact on PA counties were analyzed, and effective seasonal timing for Abies Phytophthora root rot inspection services was determined based on resulting data. Rice proteins targeted by Magnaporthe oryzae Nep1-like protein and zinc finger effectors have been further characterized by Yang lab via molecular, biochemical and genetic analyses. The host proteins targeted by them were found to play important role in protein degradation and chromatin reconfiguration and may positively or negatively mediate host resistance to M. oryzae infection. Besides the early host perception of fungal effectors, downstream signaling events such as protein phosphorylation and ubiquitination were also elucidated for their role in rice biotic and abiotic stress tolerance. To translation basic knowledge into agricultural productivity via biotechnology, the newly developed RNA-guided genome editing technology has been adapted and improved to facilitate precision breeding and genetic improvement of agronomic traits. Since rice is one of the most important crops in the world, our basic and applied research for improving disease resistance should help increase rice yield and quality, thus contributing to global food security.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Short, D.P.G., ODonnell, K., and Geiser, D.M. 2014. Clonality, recombination, and hybridization in the plumbing-inhabiting human pathogen Fusarium keratoplasticum inferred from multilocus sequence typing. BMC Evolutionary Biology 14:91.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Kang, S. 2014. Plant Pathology 2.0. Molecular Plant Pathology 15: 315318.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Xie, K., Zhang, J. and Yang, Y. 2014. Genome-wide prediction of highly specific guide RNA spacers for the CRISPR-Cas9 mediated genome editing in model plants and major crops. Mol. Plant 7: 923-926.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Xie, K. and Yang, Y. 2013. RNA-guided genome editing in plants using a CRISPR-Cas system. Mol. Plant 6:1975-1983.


Progress 07/01/13 to 09/30/13

Outputs
Target Audience: We are collaborating with USDA/ARS laboratories and international institutions to study the phylogenetics of fungal pathogens and molecular mechanism of plant disease resistance and to exchange research materials, data and protocols. The fungal plant pathogen database will assist researchers and extension educators in land grant institutions as well as researchers and regulators in state and federal agencies, such as Pennsylvania Department of Agriculture and USDA/APHIS and in diagnosis of important diseases such as sudden oak death and implementing appropriate regulatory measures. The Fusarium and Phytophthora collections and databases are also widely used by many disease diagnosticians in the U.S. and around the world. In addition, seeds companies, extension agents and growers are interested in the knowledge, materials and techniques generated by this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The work on fusaria associated with ambrosia beetles was part of the PhD thesis for Matt Kasson, giving him an opportunity to learn about how to apply phylogenetic analysis to understand the likely origin of an emerging disease problem. In addition, a few other graduate students participated in this project. How have the results been disseminated to communities of interest? The results were published in Fungal Genetics and Biology (Kasson et al. 2013 56:147-147) and were also presented during the 2013 APS meeting in Austin, TX. What do you plan to do during the next reporting period to accomplish the goals? We have several ongoing projects characterizing agriculturally important Fusarium species that will move forward in the coming year. We will also characterize the identity and diversity of Phytophthora and Pythium species cultured from streams around 13 eastern states.

Impacts
What was accomplished under these goals? Since the project was initiated 01 July, 2013, we published an article describing the diversity of fusaria associated with ambrosia beetles. These fungi are cultivated by their insect hosts as a food source, but in recent years they have emerged as an important pathogen of avocado in many parts of the world. The beetles and their Fusarium symbionts appeared in the Los Angeles basin in 2011, and have caused considerable damage to a wide range of tree hosts in the Huntington Gardens and Los Angeles Arboretum. These results fall under objectives 2 and 3, “Produce an updated molecular phylogeny of the genus Fusarium” and “Facilitate the characterization of novel and emerging Fusarium and Phytophthora pathogens…”

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Kasson, M.P., ODonnell, K., Rooney, A.P., Sink, S., Ploetz, R.C., Ploetz, J.N., Konkol, J.L., Carrillo, D., Freeman, S., Mendel, Z., Smith, J.A., Black, A.W., Hulcr, J., Bateman, C., Stefkova, K., Campbell, P.R., Geering, A.D.W., Dann, E.K., Esklalen, A., Mohotti, K., Short, D.P.G., Aoki, T., Fenstermacher, K.A., Davis, D.D. and Geiser, D.M. 2013. An inordinate fondness for Fusarium: Phylogenetic diversity of fusaria cultivated by ambrosia beetles in the genus Euwallacea on avocado and other plant hosts. Fungal Genetics and Biology. 56:147-147.