Publications by authors named "Jeffrey A Rollins"

29 Publications

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Molecular dissection of perithecial mating line development in , a species with a nontypical mating system featured by plus-to-minus switch and plus-minus mediated sexual enhancement.

Appl Environ Microbiol 2021 Apr 16. Epub 2021 Apr 16.

State Key Laboratory of Crop Stress Biology in Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, Shaanxi Province, 712100, China.

The genetic regulation of (Glomerella) sexual reproduction does not strictly adhere to the Ascomycota paradigm and remains poorly understood. Morphologically different but sexually compatible strain types termed 'plus' and 'minus' have been recognized but the biological and molecular distinctions between these strain types remain elusive. In this study, we characterized the sexual behaviors of a pair of plus and minus strains of with the aid of live cell nuclear-localized fluorescent protein labeling, gene expression and gene mutation analyses. We confirmed a genetically stable plus-to-minus switching phenomenon and demonstrated the presence of both cross-fertilized and self-fertilized perithecia within the mating line (perithecia cluster at the line of colony contact) between plus and minus strains. We demonstrated that pheromone signaling genes (a-factor-like and α-factor-like pheromones and their corresponding GPCR receptors) were differently expressed between vegetative hyphae of the two strains. Moreover, deletion of (a FUS/KSS1 MAPK) in the minus strain severely limited mating line formation whereas deletion of a GPCR (FGSG_05239 homolog) and two histone modification factors (, ) in the minus strain did not affect mating line development, but altered the ratio between cross-fertilization and self-fertilization within the mating line. We propose a model that mating line formation in involves enhanced protoperithecium differentiation and enhanced perithecium maturation of the minus strain mediated by both cross-fertilization and diffusive effectors. This study provides insights into mechanisms underlying the mysterious phenomenon of plus-minus mediated sexual enhancement being unique to fungi.Plus-minus regulation of sexual differentiation was reported in the early 1900s. Both plus and minus strains produce fertile perithecia in a homothallic but inefficient manner. However, when the two strain types encounter each other, efficient differentiation of fertile perithecia is triggered. The plus strain, by itself, can also generate minus ascospore progeny at high frequency. This nontypical mating system facilitates sexual reproduction and is -specific, the underlying molecular mechanisms, however, remain elusive. The current study revisits this longstanding mystery using as an experimental system. The presence of both cross-fertilized and self-fertilized perithecia within the mating line were directly evidenced by live cell imaging with fluorescent markers. Based on further gene expression and gene mutation analysis, a model explaining mating line development (plus-minus mediated sexual enhancement) is proposed. Data reported here has the potential to better understand mating and filamentous ascomycete sexual regulation.
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http://dx.doi.org/10.1128/AEM.00474-21DOI Listing
April 2021

Genome sequence of Monilinia vaccinii-corymbosi sheds light on mummy berry disease infection of blueberry and mating type.

G3 (Bethesda) 2021 Feb;11(2)

Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695, USA.

Mummy berry disease, caused by the fungal pathogen Monilinia vaccinii-corymbosi (Mvc), is one of the most economically important diseases of blueberries in North America. Mvc is capable of inducing two separate blighting stages during its life cycle. Infected fruits are rendered mummified and unmarketable. Genomic data for this pathogen is lacking, but could be useful in understanding the reproductive biology of Mvc and the mechanisms it deploys to facilitate host infection. In this study, PacBio sequencing and Hi-C interaction data were utilized to create a chromosome-scale reference genome for Mvc. The genome comprises nine chromosomes with a total length of 30 Mb, an N50 length of 4.06 Mb, and an average 413X sequence coverage. A total of 9399 gene models were predicted and annotated, and BUSCO analysis revealed that 98% of 1,438 searched conserved eukaryotic genes were present in the predicted gene set. Potential effectors were identified, and the mating-type (MAT) locus was characterized. Biotrophic effectors allow the pathogen to avoid recognition by the host plant and evade or mitigate host defense responses during the early stages of fruit infection. Following locule colonization, necrotizing effectors promote the mummification of host tissues. Potential biotrophic effectors utilized by Mvc include chorismate mutase for reducing host salicylate and necrotrophic effectors include necrosis-inducing proteins and hydrolytic enzymes for macerating host tissue. The MAT locus sequences indicate the potential for homothallism in the reference genome, but a deletion allele of the MAT locus, characterized in a second isolate, indicates heterothallism. Further research is needed to verify the roles of individual effectors in virulence and to determine the role of the MAT locus in outcrossing and population genotypic diversity.
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http://dx.doi.org/10.1093/g3journal/jkaa052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8022979PMC
February 2021

The infection cushion of Botrytis cinerea: a fungal 'weapon' of plant-biomass destruction.

Environ Microbiol 2021 Feb 4. Epub 2021 Feb 4.

Univ Lyon, Université Lyon 1, CNRS, INSA-Lyon, Microbiologie, Adaptation et Pathogénie, UMR 5240 MAP, 10 Rue Raphaël Dubois, Villeurbanne, F-69622, France.

The necrotrophic plant-pathogen fungus Botrytis cinerea produces multicellular appressoria dedicated to plant penetration, named infection cushions (IC). A microarray analysis was performed to identify genes upregulated in mature IC. The expression data were validated by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and biochemical assays. 1231 upregulated genes and 79 up-accumulated proteins were identified. The data support the secretion of effectors by IC: phytotoxins, ROS, proteases, cutinases, plant cell wall-degrading enzymes and plant cell death-inducing proteins. Parallel upregulation of sugar transport and sugar catabolism-encoding genes would indicate a role of IC in nutrition. The data also reveal a substantial remodelling of the IC cell wall and suggest a role for melanin and chitosan in IC function. Lastly, mutagenesis of two upregulated genes in IC identified secreted fasciclin-like proteins as actors in the pathogenesis of B. cinerea. These results support the role of IC in plant penetration and also introduce other unexpected functions for this fungal organ, in colonization, necrotrophy and nutrition of the pathogen.
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http://dx.doi.org/10.1111/1462-2920.15416DOI Listing
February 2021

Genomic and transcriptomic insights into Raffaelea lauricola pathogenesis.

BMC Genomics 2020 Aug 20;21(1):570. Epub 2020 Aug 20.

Department of Plant Pathology, University of Florida, 1453 Fifield Hall, Gainesville, FL, 32611-0680, USA.

Background: Laurel wilt caused by Raffaelea lauricola is a lethal vascular disease of North American members of the Lauraceae plant family. This fungus and its primary ambrosia beetle vector Xyleborus glabratus originated from Asia; however, there is no report of laurel wilt causing widespread mortality on native Lauraceae trees in Asia. To gain insight into why R. lauricola is a tree-killing plant pathogen in North America, we generated and compared high quality draft genome assemblies of R. lauricola and its closely related non-pathogenic species R. aguacate.

Results: Relative to R. aguacate, the R. lauricola genome uniquely encodes several small-secreted proteins that are associated with virulence in other pathogens and is enriched in secondary metabolite biosynthetic clusters, particularly polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS) and PKS-NRPS anchored gene clusters. The two species also exhibit significant differences in secreted proteins including CAZymes that are associated with polysaccharide binding including the chitin binding CBM50 (LysM) domain. Transcriptomic comparisons of inoculated redbay trees and in vitro-grown fungal cultures further revealed a number of secreted protein genes, secondary metabolite clusters and alternative sulfur uptake and assimilation pathways that are coordinately up-regulated during infection.

Conclusions: Through these comparative analyses we have identified potential adaptations of R. lauricola that may enable it to colonize and cause disease on susceptible hosts. How these adaptations have interacted with co-evolved hosts in Asia, where little to no disease occurs, and non-co-evolved hosts in North America, where lethal wilt occurs, requires additional functional analysis of genes and pathways.
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http://dx.doi.org/10.1186/s12864-020-06988-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441637PMC
August 2020

Highly Contiguous Genome Resource of Generated Using Long-Read Sequencing.

Mol Plant Microbe Interact 2020 Jun 29;33(6):790-793. Epub 2020 Apr 29.

State Key Laboratory of Crop Stress Biology in Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, 712100, Shaanxi Province, China.

is a plant-pathogenic fungus with a broad host range. It causes significant losses to important crops, including apple, pear, strawberry, and other Rosaceae and non-Rosaceae species. To date, two short read-based genomes are publicly available, but both are fragmented. In this study, we re-sequenced the genome of using nanopore long-read technology and refined the assembly with Hi-C map data. The resulting high-quality assembly is an important resource for further comparative and experimental studies with .
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http://dx.doi.org/10.1094/MPMI-11-19-0316-ADOI Listing
June 2020

The Formaldehyde Dehydrogenase SsFdh1 Is Regulated by and Functionally Cooperates with the GATA Transcription Factor SsNsd1 in Sclerotinia sclerotiorum.

mSystems 2019 Sep 10;4(5). Epub 2019 Sep 10.

College of Plant Sciences, Jilin University, Changchun, China

GATA transcription factors (TFs) are common eukaryotic regulators, and glutathione-dependent formaldehyde dehydrogenases (GD-FDH) are ubiquitous enzymes with formaldehyde detoxification activity. In this study, the formaldehyde dehydrogenase Fdh1 (SsFdh1) was first characterized as an interacting partner of a GATA TF, SsNsd1, in Genetic analysis reveals that SsFdh1 functions in formaldehyde detoxification, nitrogen metabolism, sclerotium development, and pathogenicity. Both SsNsd1 and SsFdh1 harbor typical zinc finger motifs with conserved cysteine residues. SsNsd1 regulates SsFdh1 in two distinct manners. SsNsd1 directly binds to GATA-box DNA in the promoter region of ; SsNsd1 associates with SsFdh1 through disulfide bonds formed by conserved Cys residues. The SsNsd1-SsFdh1 interaction and nuclear translocation were found to prevent efficient binding of SsNsd1 to GATA-box DNA. Site-directed point mutation of these Cys residues influences the SsNsd1-SsFdh1 interaction and SsNsd1 DNA binding capacity. SsFdh1 is regulated by and functions jointly with the SsNsd1 factor, providing new insights into the complex transcriptional regulatory mechanisms of GATA factors. is a pathogenic fungus with sclerotium and infection cushion development, making one of the most challenging agricultural pathogens with no effective control method. We identified important sclerotium and compound appressorium formation determinants, SsNsd1 and SsFdh1, and investigated their regulatory mechanism at the molecular level. SsNsd1 and SsFdh1 are zinc finger motif-containing proteins and associate with each other in the nucleus. On other hand, SsNsd1, as a GATA transcription factor, directly binds to GATA-box DNA in the promoter region of The SsNsd1-SsFdh1 interaction and nuclear translocation were found to prevent efficient binding of SsNsd1 to GATA-box DNA. Our results provide insights into the role of the GATA transcription factor and its regulation of formaldehyde dehydrogenase in stress resistance, fungal sclerotium and compound appressorium development, and pathogenicity.
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http://dx.doi.org/10.1128/mSystems.00397-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739101PMC
September 2019

Mechanisms of Broad Host Range Necrotrophic Pathogenesis in Sclerotinia sclerotiorum.

Phytopathology 2018 10 30;108(10):1128-1140. Epub 2018 Aug 30.

First author: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University; and second author: Department of Plant Pathology, University of Florida, P.O. Box 110680, Gainesville 32611-0680.

Among necrotrophic fungi, Sclerotinia sclerotiorum is remarkable for its extremely broad host range and for its aggressive host tissue colonization. With full genome sequencing, transcriptomic analyses and the increasing pace of functional gene characterization, the factors underlying the basis of this broad host range necrotrophic pathogenesis are now being elucidated at a greater pace. Among these, genes have been characterized that are required for infection via compound appressoria in addition to genes associated with colonization that regulate oxalic acid (OA) production and OA catabolism. Moreover, virulence-related secretory proteins have been identified, among which are candidates for manipulating host activities apoplastically and cytoplasmically. Coupled with these mechanistic studies, cytological observations of the colonization process have blurred the heretofore clear-cut biotroph versus necrotroph boundary. In this review, we reexamine the cytology of S. sclerotiorum infection and put more recent molecular and genomic data into the context of this cytology. We propose a two-phase infection model in which the pathogen first evades, counteracts and subverts host basal defense reactions prior to killing and degrading host cells. Spatially, the pathogen may achieve this via the production of compatibility factors/effectors in compound appressoria, bulbous subcuticular hyphae, and primary invasive hyphae. By examining the nuances of this interaction, we hope to illuminate new classes of factors as targets to improve our understanding of broad host range necrotrophic pathogens and provide the basis for understanding corresponding host resistance.
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http://dx.doi.org/10.1094/PHYTO-06-18-0197-RVWDOI Listing
October 2018

Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen Sclerotinia sclerotiorum.

mBio 2018 06 26;9(3). Epub 2018 Jun 26.

Department of Plant Pathology, University of Florida, Gainesville, Florida, USA

The necrotrophic fungal plant pathogen is responsible for substantial global crop losses annually resulting in localized food insecurity and loss of livelihood. Understanding the basis of this broad-host-range and aggressive pathogenicity is hampered by the quantitative nature of both host resistance and pathogen virulence. To improve this understanding, methods for efficient functional gene characterization that build upon the existing complete genome sequence are needed. Here, we report on the development of a clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9)-mediated strategy for creating gene disruption mutants and the application of this technique for exploring roles of known and hypothesized virulence factors. A key finding of this research is that transformation with a circular plasmid encoding Cas9, target single guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted, insertional gene mutation. We observed that 100% of the mutants integrated large rearranged segments of the transforming plasmid at the target site facilitated by the nonhomologous end joining (NHEJ) repair pathway. This result was confirmed in multiple target sites within the same gene in three independent wild-type isolates of and in a second independent gene. Targeting the previously characterized gene allowed us to confirm the loss-of-function nature of the CRISPR-Cas9-mediated mutants and explore new aspects of the mutant phenotype. Applying this technology to create mutations in a second previously uncharacterized gene allowed us to determine the requirement for melanin accumulation in infection structure development and function. Fungi that cause plant diseases by rotting or blighting host tissue with limited specificity remain among the most difficult to control. This is largely due to the quantitative nature of host resistance and a limited understanding of fungal pathogenicity. A mechanistic understanding of pathogenicity requires the ability to manipulate candidate virulence genes to test hypotheses regarding their roles in disease development. is among the most notorious of these so-called broad-host-range necrotrophic plant pathogens. The work described here provides a new method for rapidly constructing gene disruption vectors to create gene mutations with high efficiency compared with existing methods. Applying this method to characterize gene functions in , we confirm the requirement for oxalic acid production as a virulence factor in multiple isolates of the fungus and demonstrate that melanin accumulation is not required for infection. Using this approach, the pace of functional gene characterization and the understanding of pathogenicity and related disease resistance will increase.
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http://dx.doi.org/10.1128/mBio.00567-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020291PMC
June 2018

Pathogenic adaptations of Colletotrichum fungi revealed by genome wide gene family evolutionary analyses.

PLoS One 2018 24;13(4):e0196303. Epub 2018 Apr 24.

State Key Laboratory of Crop Stress Biology in Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, Shaanxi Province, China.

The fungal genus Colletotrichum contains hemibiotrophic phytopathogens being highly variable in host and tissue specificities. We sequenced a C. fructicola genome (1104-7) derived from an isolate of apple in China and compared it with the reference genome (Nara_gc5) derived from an isolate of strawberry in Japan. Mauve alignment and BlastN search identified 0.62 Mb lineage-specific (LS) genomic regions in 1104-7 with a length criterion of 10 kb. Genes located within LS regions evolved more dynamically, and a strongly elevated proportion of genes were closely related to non-Colletotrichum sequences. Two LS regions, containing nine genes in total, showed features of fungus-to-fungus horizontal transfer supported by both gene order collinearity and gene phylogeny patterns. We further compared the gene content variations among 13 Colletotrichum and 11 non-Colletotrichum genomes by gene function annotation, OrthoMCL grouping and CAFE analysis. The results provided a global evolutionary picture of Colletotrichum gene families, and identified a number of strong duplication/loss events at key phylogenetic nodes, such as the contraction of the detoxification-related RTA1 family in the monocot-specializing graminicola complex and the expansions of several ammonia production-related families in the fruit-infecting gloeosporioides complex. We have also identified the acquirement of a RbsD/FucU fucose transporter from bacterium by the Colletotrichum ancestor. In sum, this study summarized the pathogenic evolutionary features of Colletotrichum fungi at multiple taxonomic levels and highlights the concept that the pathogenic successes of Colletotrichum fungi require shared as well as lineage-specific virulence factors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196303PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915685PMC
August 2018

The GATA-type IVb zinc-finger transcription factor SsNsd1 regulates asexual-sexual development and appressoria formation in Sclerotinia sclerotiorum.

Mol Plant Pathol 2018 07 1;19(7):1679-1689. Epub 2018 Feb 1.

Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA.

The sclerotium, a multicellular structure composed of the compact aggregation of vegetative hyphae, is critical for the long-term survival and sexual reproduction of the plant-pathogenic fungus Sclerotinia sclerotiorum. The development and carpogenic germination of sclerotia are regulated by integrating signals from both environmental and endogenous processes. Here, we report the regulatory functions of the S. sclerotiorum GATA-type IVb zinc-finger transcription factor SsNsd1 in these processes. SsNsd1 is orthologous to the Aspergillus nidulans NsdD (never in sexual development) and the Neurospora crassa SUB-1 (submerged protoperithecia-1) proteins. Ssnsd1 gene transcript accumulation remains relatively low, but variable, during vegetative mycelial growth and multicellular development. Ssnsd1 deletion mutants (Δnsd1-KOs) produce phialides and phialospores (spermatia) excessively in vegetative hyphae and promiscuously within the interior medulla of sclerotia. In contrast, phialospore development occurs only on the sclerotium surface in the wild-type. Loss of SsNsd1 function affects sclerotium structural integrity and disrupts ascogonia formation during conditioning for carpogenic germination. As a consequence, apothecium development is abolished. The Ssnsd1 deletion mutants are also defective in the transition from hyphae to compound appressorium formation, resulting in a loss of pathogenicity on unwounded hosts. In sum, our results demonstrate that SsNsd1 functions in a regulatory role similar to its ascomycete orthologues in regulating sexual and asexual development. Further, SsNsd1 appears to have evolved as a regulator of pre-penetration infectious development required for the successful infection of its many hosts.
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http://dx.doi.org/10.1111/mpp.12651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638148PMC
July 2018

Sexual Reproduction in the Citrus Black Spot Pathogen, Phyllosticta citricarpa.

Phytopathology 2017 06 7;107(6):732-739. Epub 2017 Apr 7.

First, second, and seventh authors: Centre for Plant Science, Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland, Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia; third author: Centre for Plant Science, QAAFI, The University of Queensland, Queensland Bioscience Precinct, St. Lucia, QLD 4072, Australia; fourth and fifth authors: Citrus Research and Education Centre, University of Florida, Lake Alfred; and sixth author: Department of Plant Pathology, University of Florida, Gainesville.

Citrus black spot (Phyllosticta citricarpa) causes fruit blemishes and premature fruit drop, resulting in significant economic losses in citrus growing areas with summer rainfall across the globe. The mating type locus of P. citricarpa has recently been characterized, revealing the heterothallic nature of this pathogen. However, insight into the occurrence of mating and the impact of completing the sexual cycle of P. citricarpa was lacking. To investigate the occurrence and impact of sexual reproduction, we developed a method to reliably, and for the first time, produce ascospores of P. citricarpa on culture media. To demonstrate meiosis during the mating process, we identified recombinant genotypes through multilocus genotyping of single ascospores. Because the process of fertilization was not well understood, we experimentally determined that fertilization of P. citricarpa occurs via spermatization. Our results demonstrate that P. citricarpa is heterothallic and requires isolates of different MAT idiomorphs to be in direct physical contact, or for spermatia to fulfill their role as male elements to fertilize the receptive organs, in order to initiate the mating process. The impact of mating on the epidemiology of citrus black spot in the field is discussed.
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http://dx.doi.org/10.1094/PHYTO-11-16-0419-RDOI Listing
June 2017

Characterization of MAT gene functions in the life cycle of Sclerotinia sclerotiorum reveals a lineage-specific MAT gene functioning in apothecium morphogenesis.

Fungal Biol 2016 09 22;120(9):1105-17. Epub 2016 Jun 22.

Department of Plant Pathology, University of Florida, Gainesville, FL 32611-0680, USA. Electronic address:

Sclerotinia sclerotiorum (Lib.) de Bary is a phytopathogenic fungus that relies on the completion of the sexual cycle to initiate aerial infections. The sexual cycle produces apothecia required for inoculum dispersal. In this study, insight into the regulation of apothecial multicellular development was pursued through functional characterization of mating-type genes. These genes are hypothesized to encode master regulatory proteins required for aspects of sexual development ranging from fertilization through fertile fruiting body development. Experimentally, loss-of-function mutants were created for the conserved core mating-type genes (MAT1-1-1, and MAT1-2-1), and the lineage-specific genes found only in S. sclerotiorum and closely related fungi (MAT1-1-5, and MAT1-2-4). The MAT1-1-1, MAT1-1-5, and MAT1-2-1 mutants are able to form ascogonia but are blocked in all aspects of apothecium development. These mutants also exhibit defects in secondary sexual characters including lower numbers of spermatia. The MAT1-2-4 mutants are delayed in carpogenic germination accompanied with altered disc morphogenesis and ascospore production. They too produce lower numbers of spermatia. All four MAT gene mutants showed alterations in the expression of putative pheromone precursor (Ppg-1) and pheromone receptor (PreA, PreB) genes. Our findings support the involvement of MAT genes in sexual fertility, gene regulation, meiosis, and morphogenesis in S. sclerotiorum.
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http://dx.doi.org/10.1016/j.funbio.2016.06.007DOI Listing
September 2016

An atypical forkhead-containing transcription factor SsFKH1 is involved in sclerotial formation and is essential for pathogenicity in Sclerotinia sclerotiorum.

Mol Plant Pathol 2017 09 21;18(7):963-975. Epub 2016 Aug 21.

College of Plant Sciences, Jilin University, Changchun, 130062, China.

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen with a worldwide distribution. The sclerotia of S. sclerotiorum are pigmented multicellular structures formed from the aggregation of vegetative hyphae. These survival structures play a central role in the life and infection cycles of this pathogen. Here, we characterized an atypical forkhead (FKH)-box-containing protein, SsFKH1, involved in sclerotial development and virulence. To investigate the role of SsFkh1 in S. sclerotiorum, the partial sequence of SsFkh1 was cloned and RNA interference (RNAi)-based gene silencing was employed to alter the expression of SsFkh1. RNA-silenced mutants with significantly reduced SsFkh1 RNA levels exhibited slow hyphal growth and sclerotial developmental defects. In addition, the expression levels of a set of putative melanin biosynthesis-related laccase genes and a polyketide synthase-encoding gene were significantly down-regulated in silenced strains. Disease assays demonstrated that pathogenicity in RNAi-silenced strains was significantly compromised with the development of a smaller infection lesion on tomato leaves. Collectively, the results suggest that SsFkh1 is involved in hyphal growth, virulence and sclerotial formation in S. sclerotiorum.
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http://dx.doi.org/10.1111/mpp.12453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638265PMC
September 2017

Mating Type and Simple Sequence Repeat Markers Indicate a Clonal Population of Phyllosticta citricarpa in Florida.

Phytopathology 2016 11 26;106(11):1300-1310. Epub 2016 Jul 26.

All authors: Department of Plant Pathology, University of Florida, Gainesville, and first, second, and fifth authors: Citrus Research and Education Center, University of Florida, Lake Alfred.

Phyllosticta citricarpa, the citrus black spot pathogen, was first identified in Florida in March 2010. Subsequently, this pathogen has become established in Florida but can be easily confused with the endemic nonpathogenic citrus endophyte P. capitalensis. In this study, the mating-type (MAT) loci of P. citricarpa and P. capitalensis were identified via draft genome sequencing and were characterized at the structural and sequence levels. P. citricarpa was determined to have an idiomorphic, heterothallic MAT locus structure, whereas P. capitalensis was found to have a single MAT locus consistent with a homothallic mating system. A survey of P. citricarpa isolates from Florida revealed that only the MAT1-2 idiomorph existed in the Floridian population. In contrast, isolates collected from Australia exhibited a 1:1 ratio of MAT1-1 and MAT1-2 isolates. Development and analysis of simple sequence repeat markers revealed a single multilocus genotype (MLG) in the Floridian population (n = 70) and 11 MLG within the Australian population (n = 24). These results indicate that isolates of P. citricarpa from Florida are likely descendent from a single clonal lineage and are reproducing asexually. The disease management focus in Florida will need to be concentrated on the production and dispersal of pycnidiospores.
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http://dx.doi.org/10.1094/PHYTO-12-15-0316-RDOI Listing
November 2016

The Arabidopsis Mediator Complex Subunit16 Is a Key Component of Basal Resistance against the Necrotrophic Fungal Pathogen Sclerotinia sclerotiorum.

Plant Physiol 2015 Sep 4;169(1):856-72. Epub 2015 Jul 4.

Department of Microbiology and Cell Science (C.W., Z.M.) and Department of Plant Pathology (J.A.R.), University of Florida, Gainesville, Florida 32611;Department of Microbiology and Immunology, University of Buffalo, Buffalo, New York 14203 (J.Y., Y.S.);College of Life Science, Hubei University, Wuhan 430062, China (X.D.); andInterdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida 32601 (Y.Z.)

Although Sclerotinia sclerotiorum is a devastating necrotrophic fungal plant pathogen in agriculture, the virulence mechanisms utilized by S. sclerotiorum and the host defense mechanisms against this pathogen have not been fully understood. Here, we report that the Arabidopsis (Arabidopsis thaliana) Mediator complex subunit MED16 is a key component of basal resistance against S. sclerotiorum. Mutants of MED16 are markedly more susceptible to S. sclerotiorum than mutants of 13 other Mediator subunits, and med16 has a much stronger effect on S. sclerotiorum-induced transcriptome changes compared with med8, a mutation not altering susceptibility to S. sclerotiorum. Interestingly, med16 is also more susceptible to S. sclerotiorum than coronatine-insensitive1-1 (coi1-1), which is the most susceptible mutant reported so far. Although the jasmonic acid (JA)/ethylene (ET) defense pathway marker gene PLANT DEFENSIN1.2 (PDF1.2) cannot be induced in either med16 or coi1-1, basal transcript levels of PDF1.2 in med16 are significantly lower than in coi1-1. Furthermore, ET-induced suppression of JA-activated wound responses is compromised in med16, suggesting a role for MED16 in JA-ET cross talk. Additionally, MED16 is required for the recruitment of RNA polymerase II to PDF1.2 and OCTADECANOID-RESPONSIVE ARABIDOPSIS ETHYLENE/ETHYLENE-RESPONSIVE FACTOR59 (ORA59), two target genes of both JA/ET-mediated and the transcription factor WRKY33-activated defense pathways. Finally, MED16 is physically associated with WRKY33 in yeast and in planta, and WRKY33-activated transcription of PDF1.2 and ORA59 as well as resistance to S. sclerotiorum depends on MED16. Taken together, these results indicate that MED16 regulates resistance to S. sclerotiorum by governing both JA/ET-mediated and WRKY33-activated defense signaling in Arabidopsis.
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http://dx.doi.org/10.1104/pp.15.00351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577384PMC
September 2015

Fungal oxalate decarboxylase activity contributes to Sclerotinia sclerotiorum early infection by affecting both compound appressoria development and function.

Mol Plant Pathol 2015 Oct 15;16(8):825-36. Epub 2015 Apr 15.

Department of Plant Pathology, University of Florida, PO Box 110680, Gainesville, FL, 32611-0680, USA.

Sclerotinia sclerotiorum pathogenesis requires the accumulation of high levels of oxalic acid (OA). To better understand the factors affecting OA accumulation, two putative oxalate decarboxylase (OxDC) genes (Ss-odc1 and Ss-odc2) were characterized. Ss-odc1 transcripts exhibited significant accumulation in vegetative hyphae, apothecia, early stages of compound appressorium development and during plant colonization. Ss-odc2 transcripts, in contrast, accumulated significantly only during mid to late stages of compound appressorium development. Neither gene was induced by low pH or exogenous OA in vegetative hyphae. A loss-of-function mutant for Ss-odc1 (Δss-odc1) showed wild-type growth, morphogenesis and virulence, and was not characterized further. Δss-odc2 mutants hyperaccumulated OA in vitro, were less efficient at compound appressorium differentiation and exhibited a virulence defect which could be fully bypassed by wounding the host plant prior to inoculation. All Δss-odc2 phenotypes were restored to the wild-type by ectopic complementation. An S. sclerotiorum strain overexpressing Ss-odc2 exhibited strong OxDC, but no oxalate oxidase activity. Increasing inoculum nutrient levels increased compound appressorium development, but not penetration efficiency, of Δss-odc2 mutants. Together, these results demonstrate differing roles for S. sclerotiorum OxDCs, with Odc2 playing a significant role in host infection related to compound appressorium formation and function.
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http://dx.doi.org/10.1111/mpp.12239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638544PMC
October 2015

Oxaloacetate acetylhydrolase gene mutants of Sclerotinia sclerotiorum do not accumulate oxalic acid, but do produce limited lesions on host plants.

Mol Plant Pathol 2015 Aug 15;16(6):559-71. Epub 2014 Dec 15.

Department of Plant Pathology, University of Florida, Gainesville, FL, 32611-0680, USA.

The oxaloacetate acetylhydrolase (OAH, EC 3.7.1.1)-encoding gene Ss-oah1 was cloned and functionally characterized from Sclerotinia sclerotiorum. Ss-oah1 transcript accumulation mirrored oxalic acid (OA) accumulation with neutral pH induction dependent on the pH-responsive transcriptional regulator Ss-Pac1. Unlike previously characterized ultraviolet (UV)-induced oxalate-deficient mutants ('A' mutants) which retain the capacity to accumulate OA, gene deletion Δss-oah1 mutants did not accumulate OA in culture or during plant infection. This defect in OA accumulation was fully restored on reintroduction of the wild-type (WT) Ss-oah1 gene. The Δss-oah1 mutants were also deficient in compound appressorium and sclerotium development and exhibited a severe radial growth defect on medium buffered at neutral pH. On a variety of plant hosts, the Δss-oah1 mutants established very restricted lesions in which the infectious hyphae gradually lost viability. Cytological comparisons of WT and Δss-oah1 infections revealed low and no OA accumulation, respectively, in subcuticular hyphae. Both WT and mutant hyphae exhibited a transient association with viable host epidermal cells at the infection front. In summary, our experimental data establish a critical requirement for OAH activity in S. sclerotiorum OA biogenesis and pathogenesis, but also suggest that factors independent of OA contribute to the establishment of primary lesions.
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http://dx.doi.org/10.1111/mpp.12211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638444PMC
August 2015

Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses.

Nat Genet 2012 Sep 12;44(9):1060-5. Epub 2012 Aug 12.

Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany.

Colletotrichum species are fungal pathogens that devastate crop plants worldwide. Host infection involves the differentiation of specialized cell types that are associated with penetration, growth inside living host cells (biotrophy) and tissue destruction (necrotrophy). We report here genome and transcriptome analyses of Colletotrichum higginsianum infecting Arabidopsis thaliana and Colletotrichum graminicola infecting maize. Comparative genomics showed that both fungi have large sets of pathogenicity-related genes, but families of genes encoding secreted effectors, pectin-degrading enzymes, secondary metabolism enzymes, transporters and peptidases are expanded in C. higginsianum. Genome-wide expression profiling revealed that these genes are transcribed in successive waves that are linked to pathogenic transitions: effectors and secondary metabolism enzymes are induced before penetration and during biotrophy, whereas most hydrolases and transporters are upregulated later, at the switch to necrotrophy. Our findings show that preinvasion perception of plant-derived signals substantially reprograms fungal gene expression and indicate previously unknown functions for particular fungal cell types.
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http://dx.doi.org/10.1038/ng.2372DOI Listing
September 2012

Evidence for morphological, vegetative, genetic, and mating-type diversity in Sclerotinia homoeocarpa.

Phytopathology 2012 May;102(5):506-18

Department of Plant Pathology, University of Florida, Gainesville, FL, USA

Morphology, vegetative compatibility groups, and molecular characteristics were compared among 47 isolates of the dollar spot pathogen Sclerotinia homoeocarpa. Isolates were collected from cool- and warm-season turfgrasses in Florida and the northern United States. Mycelial pigment accumulation, substratal stromata formation, and symptom development were used to separate the collection into two distinct morphological types: a common-type (C-type) and a Floridian-type (F-type). Phylogenetic relationships estimated from ITS sequences supported the morphological typing. Identification and characterization of the S. homoeocarpa mating-type locus revealed an idiomorphic organization for both C- and F-types with nearly equal frequencies of each mating types present in both groups. These findings suggest heterothallic control of mating and indicate potential for outcrossing in both groups. Dollar spot disease of turfgrass in Florida is caused by two distinct morphological types of S. homoeocarpa which may be cryptic species. These findings could have implications for disease management.
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http://dx.doi.org/10.1094/PHYTO-06-11-0180DOI Listing
May 2012

pH modulation differs during sunflower cotyledon colonization by the two closely related necrotrophic fungi Botrytis cinerea and Sclerotinia sclerotiorum.

Mol Plant Pathol 2012 Aug 15;13(6):568-78. Epub 2011 Dec 15.

Université Lyon 1, CNRS, BAYER SAS, UMR 5240 Microbiologie, Adaptation et Pathogénie, 14 impasse Pierre Baizet, BP 99163, F-69263 Lyon cedex 09, France.

During pathogenesis on sunflower cotyledons, Botrytis cinerea and Sclerotinia sclerotiorum show a striking resemblance in symptom development. Based on pH change profiles, the colonization process of both fungi can be divided into two stages. The first stage is associated with a pH decrease, resulting from an accumulation of citric and succinic acids. The second stage is correlated with a pH increase, resulting from an accumulation of ammonia. In this article, we also report that oxalic acid is produced at the late stage of the colonization process and that ammonia accumulation is concomitant with a decrease in free amino acids in decaying tissues. Sclerotinia sclerotiorum produces eight-fold more oxalic acid and two-fold less ammonia than B. cinerea. Consequently, during sunflower cotyledon colonization by B. cinerea, pH dynamics differ significantly from those of S. sclerotiorum. In vitro assays support the in planta results and show that decreases in pH are linked to glucose consumption. At different stages of the colonization process, expression profiles of genes encoding secreted proteases were investigated. This analysis highlights that the expression levels of the B. cinerea protease genes are higher than those of S. sclerotiorum. This work suggests that the overt similarities of S. sclerotiorum and B. cinerea symptom development have probably masked our recognition of the dynamic and potentially different metabolic pathways active during host colonization by these two necrotrophic fungi.
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http://dx.doi.org/10.1111/j.1364-3703.2011.00772.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638627PMC
August 2012

Sclerotinia sclerotiorum γ-glutamyl transpeptidase (Ss-Ggt1) is required for regulating glutathione accumulation and development of sclerotia and compound appressoria.

Mol Plant Microbe Interact 2012 Mar;25(3):412-20

Department of Molecular Genetics and Microbiogical, University of Florida, Gainesville 32611, USA.

Transcripts encoding Sclerotinia sclerotiorum γ-glutamyl transpeptidase (Ss-Ggt1) were found to accumulate specifically during sclerotium, apothecium, and compound appressorium development in S. sclerotiorum. To determine the requirement of this protein in these developmental processes, gene deletion mutants of Ss-ggt1 were generated and five independent homokaryotic ΔSs-ggt1 mutants were characterized. All deletion mutants overproduced sclerotial initials that were arrested in further development or eventually produced sclerotia with aberrant rind layers. During incubation for carpogenic germination, these sclerotia decayed and failed to produce apothecia. Total glutathione accumulation was approximately 10-fold higher and H(2)O(2) hyperaccumulated in ΔSs-ggt1 sclerotia compared with the wild type. Production of compound appressoria was also negatively affected. On host plants, these mutants exhibited a defect in infection efficiency and a delay in initial symptom development unless the host tissue was wounded prior to inoculation. These results suggest that Ss-Ggt1 is the primary enzyme involved in glutathione recycling during these key developmental stages of the S. sclerotiorum life cycle but Ss-Ggt1 is not required for host colonization and symptom development. The accumulation of oxidized glutathione is hypothesized to negatively impact these developmental processes by disrupting the dynamic redox environment associated with multicellular development.
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http://dx.doi.org/10.1094/MPMI-06-11-0159DOI Listing
March 2012

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea.

PLoS Genet 2011 Aug 18;7(8):e1002230. Epub 2011 Aug 18.

Unité de Recherche Génomique - Info, UR1164, INRA, Versailles, France.

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.
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http://dx.doi.org/10.1371/journal.pgen.1002230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158057PMC
August 2011

The development-specific ssp1 and ssp2 genes of Sclerotinia sclerotiorum encode lectins with distinct yet compensatory regulation.

Fungal Genet Biol 2010 Jun 27;47(6):531-8. Epub 2010 Mar 27.

Department of Plant Pathology, University of Florida, Gainesville, FL 32611-0680, USA.

The Ssp1 development-specific protein is the most abundant soluble protein in sclerotia and apothecia of Sclerotinia sclerotiorum. Although closely associated with these developmental stages, the functions of the Ssp1 protein and its paralog, Ssp2, are not known. In this study, protein structure prediction analysis revealed that Ssp1 and Ssp2 are structurally similar to fucose-specific lectins. In an effort to understand the function of these abundant, development-specific proteins, a homokaryotic ssp1 deletion mutant was generated. The resulting mutant (Deltassp1) displays a wild-type growth and development phenotype in culture but produces approximately 50% fewer sclerotia in cultures supplemented with hygromycin. Genetic complementation with a wild-type copy of ssp1 restores normal sclerotium formation in the presence of hygromycin. This suggests that Ssp1 might play a role in resistance to glycoside-containing antibiotics encountered in the environment. Although a slight delay in carpogenic germination was observed, no additional effects of ssp1 loss-of-function were found in regards to apothecial morphology or fecundity. When the expression of ssp2 was examined in the Deltassp1 mutant, it was found to be expressed earlier in sclerotial development and its encoded protein accumulated to higher levels in both sclerotia and apothecia. These findings suggest regulatory compensation for loss of Ssp1 coupled with potential functional redundancy among lectins accumulating in sclerotia and apothecia.
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http://dx.doi.org/10.1016/j.fgb.2010.03.008DOI Listing
June 2010

The development-specific protein (Ssp1) from Sclerotinia sclerotiorum is encoded by a novel gene expressed exclusively in sclerotium tissues.

Mycologia 2009 Jan-Feb;101(1):34-43

Department of Plant Pathology, University of Florida, Gainesville, Florida 32611, USA.

The gene encoding a development-specific protein (Ssp1) was identified; it previously was described as the major protein present in mature sclerotia of Sclerotinia sclerotiorum. To determine the developmental specificity of ssp1 gene expression in relation to protein accumulation we examined transcript and protein accumulation during various growth and development stages of the lifecycle. We found that ssp1 transcript accumulated exclusively within developing sclerotium tissue and not in any other examined stage of growth or development. In contrast high levels of Sspl protein were detectable by western blot and tandem mass spectrometry analyses in all stages of sclerotium as well as apothecium development. Immunolocalization further indicated that Ssp1 protein bodies were depleted from the sclerotium tissue surrounding the site of apothecium germination, but by this method Sspl was not detected in the apothecium. Together these findings suggest that Sspl is not metabolized during carpogenic germination, instead it is translocated from the sclerotium to the apothecium in an antigenically novel form. Outside the Sclerotiniaceae ssp1 homologs were found only from the sclerotium-forming Aspergillus species A. flavus and A. oryzae. Further studies concerning the regulation and function of this gene and its occurrence in other species have the potential to inform our understanding of sclerotium development and the evolution of sclerotia and other forms of fungal stroma.
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http://dx.doi.org/10.3852/08-114DOI Listing
April 2009

A CRY-DASH-type photolyase/cryptochrome from Sclerotinia sclerotiorum mediates minor UV-A-specific effects on development.

Fungal Genet Biol 2008 Sep 27;45(9):1265-76. Epub 2008 Jun 27.

Department of Plant Pathology, University of Florida, 1453 Fifield Hall, Gainesville, FL 32611-0680, USA.

Apothecial development is the multicellular, sexual reproduction phase in the developmental life cycle of Sclerotinia sclerotiorum. This development begins within the sclerotium, a compact aggregation of vegetative hyphae contained within a melanized rind layer. Upon germination from the sclerotium, the apothecial stipe requires exposure to UV-A wavelengths of light to develop a fertile disc. We have identified a gene, cry1 from S. sclerotiorum that is most closely related to photolyase/cryptochrome proteins in the CRY-DASH family. We characterized this CRY-DASH ortholog from S. sclerotiorum and observed significant transcript accumulation only after exposure to UV-A and not in response to other wavelengths of light. Tissue-specific expression studies revealed that cry1 transcripts accumulate to low levels in vegetative mycelia and to higher levels in all light-exposed stages of apothecia development. Maximal cry1 transcript accumulation occurs in stipes between 2 and 6h of continuous UV-A exposure. Mutant strains carrying a deletion of cry1 exhibited a decrease in sclerotial mass and displayed greater numbers of pigmented hyphal projections on apothecial stipes under UV-A treatment but are otherwise developmentally normal. Tissue level localization of Cry1-GFP protein accumulation expressed from the native cry1 promoter was consistent with transcript localization. This study suggests that cry1 may have a function during UV exposure but is not essential for completing the developmental life cycle under laboratory conditions.
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http://dx.doi.org/10.1016/j.fgb.2008.06.004DOI Listing
September 2008

An activating mutation of the Sclerotinia sclerotiorum pac1 gene increases oxalic acid production at low pH but decreases virulence.

Mol Plant Pathol 2007 Sep;8(5):611-22

Department of Plant Pathology, University of Florida, 1453 Fifield Hall, PO Box 110680, Gainesville, FL 326110680, USA.

SUMMARY The production of oxalic acid by Sclerotinia sclerotiorum is regulated by the ambient pH environment. This regulation and that of a few investigated pH-responsive genes is mediated in part by the zinc finger transcription factor encoded by pac1, an orthologue of the Aspergillus nidulans pacC gene. We manipulated the pac1 sequence by site-directed mutagenesis to create a dominant activating pac1(c) mutation and introduced this allele into a pac1 loss-of-function (Deltapac1) strain. Consistent with a constitutive activation of Pac1 function, oxalic acid accumulation in recovered Deltapac1-pac1(c) strains was largely independent of ambient pH. Likewise, all three Deltapac1-pac1(c) strains accumulated detectable pac1 transcripts in a pH 3 environment; however, accumulation of pac1 transcripts remained alkaline-inducible, but much reduced relative to wild-type in two of the three Deltapac1-pac1(c) strains. Surprisingly, the accumulation of pg1 and acp1 transcripts, normally favoured by low pH conditions, were up-regulated across the range of ambient pH conditions examined (pH 3.4-7.2). Accumulation of neutral pH-expressed endopolygalacturonase-6 (pg6) transcripts, however, did not differ from wild-type. In pathogenicity assays using Arabidopsis and detached tomato leaflets, Deltapac1-pac1(c) strains were reduced in virulence despite the ability to accumulate oxalic acid independent of the prevailing ambient pH environment. These results support the hypothesis that appropriate gene regulation in response to ambient pH is important for full S. sclerotiorum virulence independent of oxalic acid accumulation.
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http://dx.doi.org/10.1111/j.1364-3703.2007.00423.xDOI Listing
September 2007

Deletion of the adenylate cyclase (sac1) gene affects multiple developmental pathways and pathogenicity in Sclerotinia sclerotiorum.

Fungal Genet Biol 2007 Jun 18;44(6):521-30. Epub 2006 Dec 18.

Department of Plant Pathology, University of Florida, Gainesville, FL 32611-0680, USA.

Sclerotinia sclerotiorum, a broad host range plant pathogen, produces pigmented, multihyphal sclerotia that are capable of long-term survival. Under favorable conditions, sclerotia carpogenically germinate to give rise to apothecia and forcibly discharged ascospores which serve as the primary source of inoculum in the disease cycle. The molecular regulator(s) of sclerotial development in filamentous fungi are largely unknown; however, pharmacological data has revealed that cyclic AMP (cAMP) negatively regulates sclerotial biogenesis in S. sclerotiorum. Based on this observation, we analyzed the role of cAMP by deleting the single copy adenylate cyclase (AC) sac1 gene from S. sclerotiorum. In culture, cyclic AMP levels in the knock-out (KO1) strain were greatly reduced compared to wild type, the hyphal branching pattern was altered, microconidia (spermatia) were more abundant, and aberrant sclerotia were produced in a concentric pattern. The KO1 strain was pathogenic on mechanically wounded tissues; however, virulence was severely attenuated. The pathogenicity defect on unwounded leaves is attributed to the absence of infection cushions and the attenuated virulence on wounded leaves correlates with the slow growth rate observed in culture. This study presents the first description of an adenylate cyclase mutant that affects both pathogenicity and sclerotial development in a broad host range necrotroph.
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http://dx.doi.org/10.1016/j.fgb.2006.11.005DOI Listing
June 2007

A gene with domains related to transcription regulation is required for pathogenicity in Colletotrichum acutatum causing Key lime anthracnose.

Mol Plant Pathol 2005 Sep;6(5):513-25

Citrus Research and Education Center, Institute of Food and Agricultural Sciences (IFAS), University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, USA.

SUMMARY Colletotrichum acutatum causes Key lime anthracnose (KLA) and postbloom fruit drop (PFD) of citrus. We utilized restriction enzyme-mediated integration (REMI) mutagenesis to produce six non-pathogenic mutants from a KLA isolate after screening 1064 transformants on detached Key lime leaves. Subsequently, a gene designated KLAP1 (Key Lime Anthracnose Pathogenicity) was identified from one of the mutants and was demonstrated genetically to be required for pathogenicity to Key lime leaves. The predicted polypeptide encoded by KLAP1 contains a cAMP and cGMP-dependent protein kinase phosphorylation site, and two RGD (Arg-Gly-Asp) cell attachment sequences, a bipartite nuclear targeting sequence, a fungal G-protein alpha subunit signature, a putative metal-binding zinc finger (Cys(2)His(2)) and a putative HMG-I/Y ('high mobility group' non-histone chromatin protein encoding genes) DNA-binding domain (A+T hook), suggesting that KLAP1 may function as a transcription activator in C. acutatum. Sequences homologous to KLAP1 were detected in most C. acutatum isolates examined, and similarity was found in several classes of fungi, animals, plants and bacteria, indicating that KLAP1 is a putative, uncharacterized, conserved transcription activator in fungi. Targeted gene disruption of KLAP1 yielded mutants that were blocked in the penetration stage and were completely defective in pathogenicity on Key lime leaves, but remained pathogenic to flower petals. Complementation of a klap1-null mutant with a full-length KLAP1 gene clone restored complete ability to incite lesions on Key lime. The results indicate that KLAP1 is an important pathogenicity factor in C. acutatum.
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http://dx.doi.org/10.1111/j.1364-3703.2005.00300.xDOI Listing
September 2005

The Sclerotinia sclerotiorum pac1 gene is required for sclerotial development and virulence.

Mol Plant Microbe Interact 2003 Sep;16(9):785-95

Department of Plant Pathology, 1453 Fifield Hall, University of Florida, Gainesville, FL 32611-0680, USA.

The synergistic activities of oxalic acid and endopolygalacturonases are thought to be essential for full virulence of Sclerotinia sclerotiorum and other oxalate-producing plant pathogens. Both oxalic acid production and endopolygalacturonase activity are regulated by ambient pH. Since many gene products with pH-sensitive activities are regulated by the PacC transcription factor in Aspergillus nidulans, we functionally characterized a pacC gene homolog, pac1, from S. sclerotiorum. Mutants with loss-of-function alleles of the pac1 locus were created by targeted gene replacement. In vitro mycelial growth of these pac1 mutants was normal at acidic pH, but growth was inhibited as culture medium pH was increased. Development and maturation of sclerotia in culture was also aberrant in these pac1 replacement mutants. Although oxalic acid production remained alkaline pH-responsive, the kinetics and magnitude of oxalate accumulation were dramatically altered. Additionally, maximal accumulation of endopolygalacturonase gene transcripts (pg1) was shifted to higher ambient pH. Virulence in loss-of-function pac1 mutants was dramatically reduced in infection assays with tomato and Arabidopsis. Based on these results, pac1 appears to be necessary for the appropriate regulation of physiological processes important for pathogenesis and development of S. sclerotiorum.
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http://dx.doi.org/10.1094/MPMI.2003.16.9.785DOI Listing
September 2003