Effect Of L-cysteine On The Growth Of Villosiclava Virens Mycelia And Transcriptomic Analysis

In recent years,rice false smut has become one of the main fungal diseases of rice in China.Its pathogen Villosiclava virens invades rice by mycelia and its expansion to deep rice tissues is also accomplished by mycelia.Therefore,it is very important to understand the regulation mechanism of the growth of V.virens mycelia.To explore the influence of amino acids on the growth of V.virens mycelia,20 kinds of common amino acids were supplemented to the culture media for growing V.virens mycelia,respectively.The results showed that in a certain concentration range,supplemented L-leucine and L-isoleucine could promote the growth of V.virens mycelia.However,supplemented L-tyrosine and L-cysteine inhibited the growth of V.virens mycelia.Among them,supplemented L-cysteine showed the most significant effect on the growth of V.virens mycelia.The inhibitory effect of L-cysteine on the growth of V.virens mycelia was confirmed by experiments with different culture media,V.virens strains,L-cysteine concentrations,or lighting conditions,by measuring colony diameter or mycelial biomass.In order to explore the mechanisms of inhibition by L-cysteine on the growth of V.virens mycelia,two groups of mycelial samples of ZJ09 cultured for 6 d on PSA and PSA supplemented 0.250 g·L^-1L-cysteine,respectively,were collected and used for transcriptome sequencing.2138 differentially expressed genes（DEGs）caused by L-cysteine were identified,including 985 up-regulated genes and 1153 down-regulated ones.During gene ontology（GO）enrichment analysis,1012 DEGs were enriched to the terms of biological process,cellular component and molecular function,indicating that complex mechanisms may be involved in the inhibitory effect of L-cysteine on the mycelia growth.Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis results of the DEGs indicated that influence on protein processing in endoplasmic reticulum was quite probably involved in the inhibitory effect of L-cysteine on mycelial growth.DEGs included the genes chs D,cdc6 and cdc48.The significant inhibitions of their expressions by L-cysteine were inferred to be related to its inhibitory effect on the mycelial growth.The effects of L-cysteine and lighting on the transcriptome of V.virens mycelia were compared.They induced many different DEGs,which suggested that they inhibited the mycelial growth through different mechanisms.But at the same time,L-cysteine and lighting up-regulated 534 same genes and down-regulated697 same genes,which suggested some common mechanisms for their inhibition on the mycelial growth.To further verify the results of transcriptomic analysis,CRISPR/Cas9 editing technique was applied to try to knockout the gene UV8b＿2569 of V.virens.One transformant with the knockout vector was obtained,but the gene UV8b＿2569 was not successfully knockouted.