The small GTPase MoYpt7 is required for membrane fusion in autophagy and pathogenicity of Magnaporthe oryzae

Abstract

Rab GTPases are required for vesicle-vacuolar fusion during vacuolar biogenesis in fungi. To date, little is known about the biological functions of the Rab small GTPase components in Magnaporthe oryzae. In this study, we investigated MoYpt7 of M. oryzae, a homologue of the small Ras-like GTPase Ypt7 in Saccharomyces cerevisiae. Cellular localization assays showed that MoYpt7 was predominantly localized to vacuolar membranes. Using a targeted gene disruption strategy, a ΔMoYPT7 mutant was generated that exhibited defects in mycelial growth and production of conidia. The conidia of the ΔMoYPT7 mutant were malformed and defective in the formation of appressoria. Consequently, the ΔMoYPT7 mutant failed to cause disease in rice and barley. Furthermore, the ΔMoYPT7 mutant showed impairment in autophagy, breached cell wall integrity, and higher sensitivity to both calcium and heavy metal stress. Transformants constitutively expressing an active MoYPT7 allele (MoYPT7-CA, Gln67Leu) exhibited distinct phenotypes from the ΔMoYPT7 mutant. Expression of MoYPT7-CA in MoYpt7 reduced pathogenicity and produced more appressoria-forming single-septum conidia. These results indicate that MoYPT7 is required for fungal morphogenesis, vacuole fusion, autophagy, stress resistance and pathogenicity in M. oryzae.

Autophagy-related protein MoAtg14 is involved in differentiation, development and pathogenicity in the rice blast fungus Magnaporthe oryzae

“…No autophagic bodies were observed in the vacuole lumens of the Δ Moatg14 mutant using microscopy assays. These results are in accord with the movement and proteolysis of GFP-MoAtg8 in the Δ Moatg1 mutant, in which the autophagy was fully blocked1842. It has been reported that most of autophagy-related proteins accumulate at the PAS and generate autophagosomes.…”

“…Other media included V8 medium, OMA medium and MM as reported previously42. For DNA and RNA extractions, mycelia were harvested from 7-day-old cultures grown in liquid CM at 25 °C with shaking at 150-rpm.…”

“…General molecular biology techniques for nucleic acid analysis were performed according to standard protocols51. Phenotypic analyses including screens for the formation of conidia, conidiophore formation, conidia germination, appressoria formation, and pathogenicity assays were carried out as reported previously42.…”

“…The C-terminal, N-terminal, and coiled-coil domain (CCD) fragments were amplified from the MoATG14 cDNA using the primers Atg14C-F/R, Atg14N-F/R, and Atg14CCD-F/R, respectively. The amplified fragments were inserted into the Sma I/ Xba I, Xba I, and SmaI/ Bam HI sites of the vector pKD6142 to generate the vectors pMoAtg14-ΔC, pMoAtg14-ΔN, and pMoAtg14-ΔCCD, respectively. The vectors were introduced into the mutant Δ Moatg14 via ATMT.…”

“…For the GFP-MoAtg8 proteolysis assay, the pGFP-MoAtg8 vector was transformed into Guy11 and the mutants using the ATMT method as previously described42. Transformants expressing GFP-MoAtg8 were selected to detect full-length GFP-MoAtg8 and free GFP with anti-GFP western blots.…”