Functional Analysis Of Actin-regulating Kinase MoArk1 Binding Proteins MoCAP And MoAbp1 In Development And Pathogenicity Of Magnaporthe Oryzae

Rice blast caused by Magnaporthe grisea which is one of the most destructive causal agents during rice cultivation severely threaten the yield of rice crops.The disease has occurred in the whole growth and development period of rice.Its popularity varies in different areas and different years,but each year rice blast causes losses of between 10%and 20%of the rice harvest.The disease also is one of the most destructive diseases in rice producing areas of our country,and it threatens the production of rice.At present,the use of resistant cultivars and application of fungicides is still the main control measures.However,when a new pathogenic or anti-fungicide population emerge,the newly developed disease-resistant rice cultivars are prone to lose resistance.Therefore,the rice blast is always an important problem to be solved.And understanding the development and pathogenicity-related molecular mechanisms of M.oryzae is crucial to prevent the disease.In a previous study,actin-regulating kinase Mo Ark1 displays conserved functions important in endocytosis and actin organization,and MoArkl is required for maintaining the cell wall integrity,growth,and full virulence of M.oryzae.In this paper,we focused on functional analysis of MoArkl binding proteins MoCapA,MoCapB and MoAbpl during its development and pathogenicity in M.oryzae,and the main results are showed as follows:To understand how MoArkl might function,we identified F-actin capping protein homologs from M.oryzae(MoCAP)that interact with MoArk1 in vivo.MoCAP is heterodimeric consisting of a and β subunits(MoCapA and MoCapB,respectively),and further sequence analysis showed that MoCapA contains two conserved F-actin capping motifs,whereas MoCapB contains one F-actin capping motif and single.Deletions of αor/and β subunits resulted in abnormal morphogenesis and reductions in mycelial growth and conidia formation.This may be due to the possibility that a single MoCAP subunit might be non-functioning without its heterodimeric partner.The ΔMocap mutants failed to penetrate and cause invasive growth within host cells.In addition,the ΔMocap mutants exhibited delayed endocytosis,mitosis and autophagy.Consistent with above findings,MoCAP proteins interact with Mo Act1,co-localizes with actin,and participates in actin assembly.In the transformants of which the F-actin capping motifs were deleted,GFP fluorescence appeared in the cytoplasm of conidia with no actin localization pattern shown.Defects in growth and pathogenicity were similar to those of the ΔMocap mutant.And the F-actin-capping motifs are essential for the interactions between MoCAP and Mo Act1.Mass spectrometry showed that the serine at the 85th residue of MoCapA and serine at the 285th residue of MoCapB were phosphorylated by MoArkl.Further analysis showed that MoCAP proteins functions are negatively regulated by MoArkl through protein phosphorylation.Finally,exogenous PIP2 failed to modulate actin ring formation in theΔMocap mutants,in contrast to the wild-type strain,suggesting that MoCAP may also mediate phospholipid signaling in the regulation of the actin organization.These results together demonstrated that MoCAP proteins whose functions are regulated by MoArkl and PIP2 are important for multiple cellular processes and virulence in M.oryzae and provided new insights into the actin cytoskeleton dynamics.We identified actin binding protein from M.oryaze(MoAbp1)consisting of one ADFH domain and two SH3 domains that interact with MoArk1 in vivo.And we confirmed the interactions between MoArkl and MoAbpl.Deletion of MoABPl resulted in reduction in mycelial growth and attenuated virulence.In addition,the ΔMoabpl mutant exhibited delayed endocytosis and abnormal actin morphogenesis.Further analysis showed that localization of MoArkl proteins to cortical patches was reduced dramatically and MoArk1 is not phosphorylated in ΔMoabp1.Finally,the cyclase-associated protein MoCap1 is not capable of being localized to cortical patches in the absence of MoAbp1,and the intracellular cAMP level is reduced in ΔMoabp1.MoAbp1 interacts with not only MoArk1 and MoCap1,but also MoAct1,through the conserved F-SH3,E-SH3,and ADFH domains.MoAbp1 colocalization wi0th actin patches depends on the ADFH domain,and all MoAbp1 domains are required for its normal function.Our results indicate that MoAbp1 functions as an adaptor protein linking MoArk1 and MoCap1 to MoAct1 to regulate actin cytoskeleton dynamic necessary for growth and pathogenicity.In summary,the actin cytoskeleton plays important roles in the rie blast fungus.The F-actin capping proteins MoCAP and actin binding protein MoAbpl coordinate actin dynamics to facilitate multiple celluar processes and virulence in M.oryzae.