Upon infection, phytopathogenic fungi secrete an array of hydrolytic enzymes that can degrade components of the host epidermis, including waxes, the cuticle, and cell walls. Cellulases, which can hydrolyze crystalline cellulose in the plant cell wall, are among these hydrolytic enzymes. Here, we provide RNAi-based evidence to show that cellulases belonging to glycosyl hydrolase (GH) families 6 and 7 contribute to the penetration of the host epidermis and further invasion by the phytopathogenic fungus Magnaporthe oryzae. The GH6 and GH7 cellulases likely include all members of the cellobiohydrolase family and some endoglucanases in M. oryzae. Quantitative reverse-transcriptase polymerase chain reaction analysis indicated that more than half of the cellulases were highly induced during infection. We constructed knock-down (KD) mutants of these cellulases using the building blocks method we reported previously. The transcript levels of the target genes and cellulase activity were considerably reduced in the KD mutants. The KD mutants resulted in fewer lesions, less penetration, and infection of fewer cells compared with the parent strain. Cytological analyses showed that a high rate of papilla formation blocked invasion of the KD mutants into host cells. These results suggest that the GH6 and GH7 cellulases play roles in the virulence of M. oryzae.
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Due to functional redundancy, it is often difficult to genetically analyse the biological function of fungal cell wall-degrading enzymes that belong to a gene family. To overcome this difficulty, we used RNAi to knock-down (KD) multiple xylanase genes to elucidate their roles in the pathogenicity of the blast fungus, Magnaporthe oryzae. To obtain the maximum average efficiency of gene silencing for the xylanase genes, we used the 'building blocks method', in which a 40 bp sequence was chosen from an endoxylanase gene, and 10 such sequences from 10 endoxylanases were combined to make an artificial RNAi trigger by synthetic DNA. Quantitative RT-PCR analysis revealed that the transcript levels of all the expressed xylanase genes were significantly reduced in KD mutants with the artificial RNAi trigger. Even though the KD mutants did not completely lose their pathogenicity to host plants, the number of lesions, rate of penetration and extent of infected cells were all reduced in KD mutant-infected leaves. The degree of pathogenicity reduction was associated with the silencing levels of xylanase mRNA and enzymatic activity in the KD mutants. Cytological analysis indicated that xylanases play significant roles in both vertical penetration and horizontal expansion of M. oryzae in infected plants.
