Jeffrey A. Rollins

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

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.

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

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.

pH Signaling in Sclerotinia sclerotiorum: Identification of a pacC/RIM1 Homolog

ABSTRACT Sclerotinia sclerotiorum acidifies its ambient environment by producing oxalic acid. This production of oxalic acid during plant infection has been implicated as a primary determinant of pathogenicity in this and other phytopathogenic fungi. We found that ambient pH conditions affect multiple processes in S. sclerotiorum. Exposure to increasing alkaline ambient pH increased the oxalic acid accumulation independent of carbon source, sclerotial development was favored by acidic ambient pH conditions but inhibited by neutral ambient pH, and transcripts encoding the endopolygalacturonase gene pg1 accumulated maximally under acidic culture conditions. We cloned a putative transcription factor-encoding gene, pac1, that may participate in a molecular signaling pathway for regulating gene expression in response to ambient pH. The three zinc finger domains of the predicted Pac1 protein are similar in sequence and organization to the zinc finger domains of the A. nidulans pH-responsive transcription factor PacC. The promoter of pac1 contains eight PacC consensus binding sites, suggesting that this gene, like its homologs, is autoregulated. Consistent with this suggestion, the accumulation ofpac1 transcripts paralleled increases in ambient pH. Pac1 was determined to be a functional homolog of PacC by complementation of an A. nidulans pacC-null strain with pac1. Our results suggest that ambient pH is a regulatory cue for processes linked to pathogenicity, development, and virulence and that these processes may be under the molecular regulation of a conserved pH-dependent signaling pathway analogous to that in the nonpathogenic fungus A. nidulans.

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

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.

Multi-factor regulation of pectate lyase secretion by Colletotrichum gloeosporioides pathogenic on avocado fruits

Tissue alkalinization during Colletotrichum gloeosporioides attack enhances the expression of PELB, which encodes pectate lyase (PL), and PL secretion, which is considered essential for full virulence. We studied the regulation of PL secretion by manipulation of C. gloeosporioides PELB. PELB was down-regulated by knocking out PAC1, which encodes the PacC transcription factor that regulates gene products with pH-sensitive activities. We functionally characterized a PACC gene homologue, PAC1, from C. gloeosporioides wild-type (WT) Cg-14 and two independent deletion strains, Deltapac1(372)and Deltapac1(761). Loss-of-function PAC1 mutants showed 85% reduction of PELB transcript expression, delayed PL secretion and dramatically reduced virulence, as detected in infection assays with avocado fruits. In contrast, PELB was up-regulated in the presence of carbon sources such as glucose. When glucose was used as a carbon source in the medium for the WT strain and the Deltapac1 mutant at pH 6.0, PELB transcript expression and PL secretion were activated. Other sugars, such as sucrose and fructose (but not galactose), also activated PELB expression. These results suggest that the pH-regulated response is only part of a multi-factor regulation of PELB, and that sugars are also needed to promote the transition from quiescent to active necrotrophic development by the pathogen.

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

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.

Genetic analysis of cross fertility between two self-sterile strains of Glomerella graminicola.

The ascomycete Glomerella cingulata has an unusual and complex mating system which is con- trolled by multiple, multiallelic loci. Cross fertility be- tween different isolates occurs via complementation of mutated fertility genes, a process known as unbal- anced heterothallism. We have examined the herita-

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

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 Ssp1 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 Ssp1 was not detected in the apothecium. Together these findings suggest that Ssp1 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.

The Complete Genome Sequence of the Phytopathogenic Fungus Sclerotinia sclerotiorum Reveals Insights into the Genome Architecture of Broad Host Range Pathogens

Sclerotinia sclerotiorum is a phytopathogenic fungus with over 400 hosts including numerous economically important cultivated species. This contrasts many economically destructive pathogens that only exhibit a single or very few hosts. Many plant pathogens exhibit a “two-speed” genome. So described because their genomes contain alternating gene rich, repeat sparse and gene poor, repeat-rich regions. In fungi, the repeat-rich regions may be subjected to a process termed repeat-induced point mutation (RIP). Both repeat activity and RIP are thought to play a significant role in evolution of secreted virulence proteins, termed effectors. We present a complete genome sequence of S. sclerotiorum generated using Single Molecule Real-Time Sequencing technology with highly accurate annotations produced using an extensive RNA sequencing data set. We identified 70 effector candidates and have highlighted their in planta expression profiles. Furthermore, we characterized the genome architecture of S. sclerotiorum in comparison to plant pathogens that exhibit “two-speed” genomes. We show that there is a significant association between positions of secreted proteins and regions with a high RIP index in S. sclerotiorum but we did not detect a correlation between secreted protein proportion and GC content. Neither did we detect a negative correlation between CDS content and secreted protein proportion across the S. sclerotiorum genome. We conclude that S. sclerotiorum exhibits subtle signatures of enhanced mutation of secreted proteins in specific genomic compartments as a result of transposition and RIP activity. However, these signatures are not observable at the whole-genome scale.

Sclerotinia sclerotiorum γ-Glutamyl Transpeptidase (Ss-Ggt1) Is Required for Regulating Glutathione Accumulation and Development of Sclerotia and Compound Appressoria

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.