Glucosinolates accumulate mainly in cruciferous plants and their hydrolysis-derived products play important roles in plant resistance against pathogens. The pathogen Botrytis cinerea has variable sensitivity to glucosinolates, but the mechanisms by which it responds to them are mostly unknown. Exposure of B . cinerea to glucosinolate-breakdown products induces expression of the Major Facilitator Superfamily transporter, mfsG , which functions in fungitoxic compound efflux. Inoculation of B. cinerea on wild-type Arabidopsis thaliana plants induces mfsG expression to higher levels than on glucosinolate-deficient A. thaliana mutants. A B. cinerea strain lacking functional mfsG transporter is deficient in efflux ability. It accumulates more isothiocyanates (ITCs) and is therefore more sensitive to this compound in vitro ; it is also less virulent to glucosinolates-containing plants. Moreover, mfsG mediates ITC efflux in Saccharomyces cerevisiae cells, thereby conferring tolerance to ITCs in the yeast. These findings suggest that mfsG transporter is a virulence factor that increases tolerance to glucosinolates.

In September 2014, a new tobamovirus was discovered in Israel that was able to break Tm-2-mediated resistance in tomato that had lasted 55 years. The virus was isolated, and sequencing of its genome showed it to be tomato brown rugose fruit virus (ToBRFV), a new tobamovirus recently identified in Jordan. Previous studies on mutant viruses that cause resistance breaking, including Tm-2-mediated resistance, demonstrated that this phenotype had resulted from only a few mutations. Identification of important residues in resistance breakers is hindered by significant background variation, with 9-15% variability in the genomic sequences of known isolates. To understand the evolutionary path leading to the emergence of this resistance breaker, we performed a comprehensive phylogenetic analysis and genomic comparison of different tobamoviruses, followed by molecular modeling of the viral helicase. The phylogenetic location of the resistance-breaking genes was found to be among host-shifting clades, and this, together with the observation of a relatively low mutation rate, suggests that a host shift contributed to the emergence of this new virus. Our comparative genomic analysis identified twelve potential resistance-breaking mutations in the viral movement protein (MP), the primary target of the related Tm-2 resistance, and nine in its replicase. Finally, molecular modeling of the helicase enabled the identification of three additional potential resistance-breaking mutations.

SUMMARYScreening large populations for carriers of known or de novo rare single nucleotide polymorphisms (SNPs) is required both in Targeting induced local lesions in genomes (TILLING) experiments in plants and in screening of human populations. We previously suggested an approach that combines the mathematical field of compressed sensing with next-generation sequencing to allow such large-scale screening. Based on pooled measurements, this method identifies multiple carriers of heterozygous or homozygous rare alleles while using only a small fraction of resources. Its rigorous mathematical foundations allow scalable and robust detection, and provide error correction and resilience to experimental noise. Here we present a largescale experimental demonstration of our computational approach, in which we targeted a TILLING population of 1024 Sorghum bicolor lines to detect carriers of de novo SNPs whose frequency was less than 0.1%, using only 48 pools. Subsequent validation confirmed that all detected lines were indeed carriers of the predicted mutations. This novel approach provides a highly cost-effective and robust tool for biologists and breeders to allow identification of novel alleles and subsequent functional analysis.

The necrotrophic fungus Botrytis cinerea, is considered a major cause of postharvest losses in a wide range of crops. The common fungal extracellular membrane protein (CFEM), containing a conserved eight-cysteine pattern, was found exclusively in fungi. Previous studies in phytopathogenic fungi have demonstrated the role of membrane-bound and secreted CFEM-containing proteins in different aspects of fungal virulence. However, non-G protein-coupled receptor (non-GPCR) membrane CFEM proteins have not been studied yet in phytopathogenic fungi. In the present study, we have identified a non-GPCR membrane-bound CFEM-containing protein, Bcin07g03260, in the B. cinerea genome, and generated deletion mutants, ΔCFEM-Bcin07g03260, to study its potential role in physiology and virulence. Three independent ΔCFEM-Bcin07g03260 mutants showed significantly reduced progression of a necrotic lesion on tomato (Solanum lycopersicum) leaves. Further analysis of the mutants revealed significant reduction (approximately 20–30%) in conidial germination and consequent germ tube elongation compared with the WT. Our data complements a previous study of secreted ΔCFEM1 mutants of B. cinerea that showed reduced progression of necrotic lesions on leaves, without effect on germination. Considering various functions identified for CFEM proteins in fungal virulence, our work illustrates a potential new role for a non-GPCR membrane CFEM in pathogenic fungi to control virulence in the fungus B. cinerea.