Studies On The Function Of Authophagy Protein UvATG6 And UPR Transcription Factor UvHAC1 In Ustilaginoidea Virens

Rice false smut is a rice panicle disease caused by Ustilaginoidea virens,which is distributed all over the world.Autophagy and unfolded protein response(UPR)are important in the circulation of intracellular substance and play important roles in maintaining cell homeostasis and normal physiological function.In this study,UvATG6 involved in autophagy and UvHAC1 involved in UPR processes were identified,and their functions and mechanisms for the growth,development,and pathogenicity of U.virens were further explored.UvATG6 is an autophagy related protein in U.virens and contains a conserved APG6 domain.The expression level of UvATG6 gradually increased and reached the highest level at 5 dpi.UvATG6 was knocked out by combining CRISPR/Cas9 with homologous recombination.The expression of autophagy related genes in UvATG6 mutants was significantly reduced,and autophagy function was severely impaired,which could not be performed normally.The growth of UvATG6 knockout mutants was significantly decreased.Conidiation of UvATG6 mutants was almost abolished,and autophagy occurred during conidial germination.UvATG6 was involved in abiotic stress tolerance.The tolerance of UvATG6 mutants to osmotic and cell wall stress was significantly decreased,but the tolerance to reactive oxygen stress was significantly increased.In addition,UvATG6 mutants could not produce rice smut balls after inoculation,indicating that UvATG6 plays an important role in the pathogenic process of U.virens.UvATG6 interacted with two Bax inhibitor proteins Uv BI-1 and Uv BI-1b of U.virens and suppressed Bax induced cell death.UvHAC1 is a transcription factor of UPR pathway of U.virens.The expression level of UvHAC1 reached the highest at 3 dpi,which was 80 times higher than that at mycelium stage.UvHAC1 has two transcripts under different conditions.After induction by DTT,UvHAC1 generates frameshift mutation through 20 bp alterative splicing,forming a shorter transcript with transcriptional activation.The growth of UvHAC1 knockout mutants decreased and they lost the ability to produce conidia,and thus the pathogenicity of UvHAC1 mutants was completely lost.UvHAC1 was involved in the tolerance to various abiotic stresses,and tolerance to osmotic and cell wall stress was significantly decreased,but tolerance to reactive oxygen stress was significantly increased.This study revealed that UvATG6 and UvHAC1 were involved in the protein turnover pathways in U.virens.Autophagy or UPR was significantly inhibited after knocking out UvATG6 and UvHAC1,respectively.In addition,the expressions of UvATG6 and UvHAC1 increased significantly during the infection stages and these two genes played indispensable roles in the conidiation and pathogenicity of U.virens.This study revealed two genes that play important functions in different physiological processes and pathogenicity of U.virens.These results may provide new gene targets for the control of rice false smut.