Ss-Sl2, a Novel Cell Wall Protein with PAN Modules, Is Essential for Sclerotial Development and Cellular Integrity of Sclerotinia sclerotiorum

The sclerotium is an important dormant body for many plant fungal pathogens. Here, we reported that a protein, named Ss-Sl2, is involved in sclerotial development of Sclerotinia sclerotiorum. Ss-Sl2 does not show significant homology with any protein of known function. Ss-Sl2 contains two putative PAN modules which were found in other proteins with diverse adhesion functions. Ss-Sl2 is a secreted protein, during the initial stage of sclerotial development, copious amounts of Ss-Sl2 are secreted and accumulated on the cell walls. The ability to maintain the cellular integrity of RNAi-mediated Ss-Sl2 silenced strains was reduced, but the hyphal growth and virulence of Ss-Sl2 silenced strains were not significantly different from the wild strain. Ss-Sl2 silenced strains could form interwoven hyphal masses at the initial stage of sclerotial development, but the interwoven hyphae could not consolidate and melanize. Hyphae in these interwoven bodies were thin-walled, and arranged loosely. Co-immunoprecipitation and yeast two-hybrid experiments showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Woronin body major protein (Hex1) and elongation factor 1-alpha interact with Ss-Sl2. GAPDH-knockdown strains showed a similar phenotype in sclerotial development as Ss-Sl2 silenced strains. Hex1-knockdown strains showed similar impairment in maintenance of hyphal integrity as Ss-Sl2 silenced strains. The results suggested that Ss-Sl2 functions in both sclerotial development and cellular integrity of S. sclerotiorum

Adenylyl Cyclase Plays a Regulatory Role in Development, Stress Resistance and Secondary Metabolism in Fusarium fujikuroi

The ascomycete fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces secondary metabolites of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. Production of these metabolites is regulated by nitrogen availability and, in a specific manner, by other environmental signals, such as light in the case of the carotenoid pathway. A complex regulatory network controlling these processes is recently emerging from the alterations of metabolite production found through the mutation of different regulatory genes. Here we show the effect of the targeted mutation of the acyA gene of F. fujikuroi, coding for adenylyl cyclase. Mutants lacking the catalytic domain of the AcyA protein showed different phenotypic alterations, including reduced growth, enhanced production of unidentified red pigments, reduced production of gibberellins and partially derepressed carotenoid biosynthesis in the dark. The phenotype differs in some aspects from that of similar mutants of the close relatives F. proliferatum and F. verticillioides: contrary to what was observed in these species, ΔacyA mutants of F. fujikuroi showed enhanced sensitivity to oxidative stress (H2O2), but no change in heavy metal resistance or in the ability to colonize tomato tissue, indicating a high versatility in the regulatory roles played by cAMP in this fungal group

Non-aqueous homogenous biocatalytic conversion of polysaccharides in ionic liquids using chemically modified glucosidase

The increasing requirement to produce platform chemicals and fuels from renewable sources means advances in biocatalysis are rapidly becoming a necessity. Biomass is widely used in nature as a source of energy and as chemical building blocks. However, recalcitrance towards traditional chemical processes and solvents provides a significant barrier to widespread utility. Here, by optimizing enzyme solubility in ionic liquids, we have discovered solvent-induced substrate promiscuity of glucosidase, demonstrating an unprecedented example of homogeneous enzyme bioprocessing of cellulose. Specifically, chemical modification of glucosidase for solubilization in ionic liquids can increase thermal stability to up to 137 °C, allowing for enzymatic activity 30 times greater than is possible in aqueous media. These results establish that through a synergistic combination of chemical biology (enzyme modification) and reaction engineering (solvent choice), the biocatalytic capability of enzymes can be intensified: a key step towards the full-scale deployment of industrial biocatalysis

Virulence differences among Sclerotinia sclerotiorum isolates determines host cotyledon resistance responses in Brassicaceae genotypes

Differences in Sclerotinia rot (SR) disease severity, caused by two categorized pathotypes and one more recent isolate of S. sclerotiorum and measured in terms of cotyledon lesion diameter, were studied across diverse Brassicaceae hosts to characterize host response and pathogen virulence. There were significant differences (P =0.001) between genotypes, isolates and a significant genotype x isolate interaction. The mean diameter of cotyledon lesions ranged from 5 mm in the most resistant genotypes (e.g., Brassica juncea Ringot I and Seeta) to = 13.6 mm in the most susceptible genotypes (e.g., B. tournefortii Wild turnip #1 and #2, Sisymbrium irio London rocket Wild #1 and #2, and B. nigra 4381). Responses, in at least one experiment for some B. juncea (e.g., Seeta, Ringot I) and Raphanus sativus (e.g., Colonel) genotypes, were generally highly resistant irrespective of the isolate used, making them ideal sources of resistance to exploit for developing new varieties with more effective resistance to SR across multiple pathotypes of this pathogen. In contrast, some other genotypes showed significant isolate dependency, with high levels of resistance against one isolate (e.g., B. napus Charlton against the WW4 isolate; B. napus Oscar against the ‘Cabbage’ isolate) but quite susceptible to other isolates (e.g., B. napus Charlton against the ‘Cabbage’ and MBRS1 isolates; B. napus Oscar against the WW4 isolate). These findings highlight the value from using pathotypes of different physiological specialization in screening programs to identify host resistance that is durable across multiple pathotypes. Distinct host resistance symptom types were reported for the first time on some genotypes against isolate WW4; including a distinct yellow halo observed around lesions on B. napus RQ001, indicative of leaf senescence involved in programmed cell death (PCD); a distinct dark brown margin observed around lesions on R. sativus, indicative of a hypersensitive response (HR); and the HR ‘flecking’ on Sinapis alba Concerta and B. juncea Seeta. That WW4 was the most pathogenic isolate for genotypes such as B. juncea Hetianyoucai and B. napus Oscar that showed high level resistance to the ‘Cabbage’ isolate and intermediate resistance to MBRS-1, dispels previously held views that WW4 was a largely avirulent pathotype of little consequence. Rather, isolate WW4 offers unique opportunities to investigate HR and PCD host resistance responses to S. sclerotiorum in Brassicaceae