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

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.

Methionine biosynthesis is essential for infection in the rice blast fungus Magnaporthe oryzae.

Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection.

Magnaporthe grisea avirulence gene ACE1 belongs to an infection-specific gene cluster involved in secondary metabolism.

The avirulence gene ACE1 from the rice blast fungus Magnaporthe grisea encodes a polyketide synthase (PKS) fused to a nonribosomal peptide synthetase (NRPS) probably involved in the biosynthesis of a secondary metabolite recognized by Pi33 resistant rice (Oryza sativa) cultivars. Analysis of the M. grisea genome revealed that ACE1 is located in a cluster of 15 genes, of which 14 are potentially involved in secondary metabolism as they encode enzymes such as a second PKS-NRPS (SYN2), two enoyl reductases (RAP1 and RAP2) and a putative Zn(II)(2)Cys(6) transcription factor (BC2). These 15 genes are specifically expressed during penetration into the host plant, defining an infection-specific gene cluster. A pORF3-GFP transcriptional fusion showed that the highly expressed ORF3 gene from the ACE1 cluster is only expressed in appressoria, as is ACE1. Phenotypic analysis of deletion or disruption mutants of SYN2 and RAP2 showed that they are not required for avirulence in Pi33 rice cultivars, unlike ACE1. Inactivation of other genes was unsuccessful because targeted gene replacement and disruption were inefficient at this locus. Overall, the ACE1 gene cluster displays an infection-specific expression pattern restricted to the penetration stage which is probably controlled at the transcriptional level and reflects regulatory networks specific to early stages of infection.