Regulation Of Conidiogenesis By COS1 In Magnaporthe Oryzae

Rice(Oryza saliva)is one of the most important global food crops,which is consumed by more than half of the world’s population.Asia is the largest production and consumption region.Rice blast is the most serious disease in rice production,causing about 10%to 30%of rice yield loss annually,which threats the world food security.Rice blast is caused by the fungus Rice leaves infected by M.oryzae display necrotic lesions and heavy infections can kill rice seedlings.In previous research,we established a high efficient transformation system of rice blast fungus by ATMT.Further investigation revealed that this mutant did not develop any conidiophore,and that the T-DNA was integrated into an annotated gene designated as conidiophore stalk-less or or COSI.Sequence analysis revealed that COSI putatively encodes a zinc-finger protein and the protein was revealed localized to nucleus.DNA sequence analysis of promoter regions of those of COS I-dependent genes showed enrichment in the DNA sequence AAAAGAAA(A4GA3),the putative COS I-binding motif.Gel shift experiments showed that COSI binds to DNA elements with A4GA3 motif.These suggest that many of the COSI-dependent transcripts may be regulated directly by COSI binding.RT-PCR-based expression analysis indicated that one gene homologous to Aspergillus nidulans flbA,RGSI(Regulators of G-protein signaling),was affected by the COSI mutation.This implies that there may be some kind of interaction relations between the two genes.RGSI is a regulatory factor in G-protein signaling pathway which regulate conidiation.Based on these research background,we studied COSI and RGSI in regulation of conidiophore development.We firstly constructed a double mutant of the two genes,COSI and RGSI,which is designated as MR.MR grows on oat culture medium with white aerial hyphae similar to the wildtype Y34.When it grows on the complete medium,the colonies display less and smoother aerial hyphae.Under the microscope,the wildtype Y34 can be observed a lot of conidium,andΔMorgsl mutant strain conidium generated in number significantly less than the wild type.Microscopic observation also found that MR does not have the ability to produce conidium.Appressorium-like structures were also observed in ΔMocosl and that maybe the reasonΔMocosl aggravate infection of wounded leaves.Inoculations of rice roots and wounded leaves with mycelia suggested that MR is not required for pathogenicity,indicating that RGS1 may have a role in some unknown mechanism of mycelial infection of M.oryzae.We then analysed expression of the conidiation-related genes in ΔMocosl、△Morgs1 and ΔMocos1/ΔMorgs1 by qRT-PCR.The results showed that when mutation of cosl down-regulated con6、brlA、flbA、ΔMocos1/ΔMorgs1 also down-regulated con6、brlA and fibA,while up-regulated con8、flbC、flbD、fluG、medA and stuA.This mutant and the deletion mutant MR are markedly deficient in melanin.Compared with wild-type Y34(1.66μg/mL),ΔMorgs1(1.6μg/mL)and ΔMocosl(1.47μg/mL)are less than wild-type.Moreover,△morgs1/ΔMocos1(0.6μg/mL)are markedly deficient in melanin.Associating with that,we propose that mutation of the COS1 and RGS1 genes may have an effect on the melanin production for vegetative growth.We then analysed expression of the melanin biosynthetic particular genes inΔMocos1、ΔMorgs1 and ΔMocos1/ΔMorgs1 by qRT-PCR.The results showed thatΔMocos1、△Morgs1 and ΔMocos1/ΔMorgs1 down-regulated GTHN and MSD.The results showed that when mutation of RGS1 down-regulated BUF1 and that maybe one of the reasons for the decrease in melanin secretion.Further gel shift assay indicate that COS1 can directly bind the A4GA3 motifs from upstream regions of the RGS1 promoter gene,suggesting that COS1 may regulate gene expression by DNA binding.