| Northern Corn Leaf Blight is one of the most destructive leaf diseases of maize caused by Setosphaeria turcica,a pathogen of hemibiotrophic fungus.It occurs widely in corn planting areas of China,with the annual incidence area of more than 60 million acres,causing great economic losses to agricultural production.Studies have shown that the extension of filamentous fungal hyphae depends on the polar growth of structures such as the spitzenkorper at its tip.Related substances are transported from the secretory vesicles gathered in the spitzenkorper to the growth points at the tip of the hyphae,so that the vesicle contents are used to synthesize the cell wall and other substances to promote the continuous growth of the hyphae and infection.The Rab protein family is the largest subfamily in the Ras superfamily of small G-protein,which exists in all eukaryotes.It acts as a molecular switch for vesicle transport between various organelles,and plays a key role during the process of growth,development and pathogenic in fungi.As an important member of the Rab family,Rab4 is an important factor,which regulating the endocytosis and vesicle circle transport.Our research group previously obtained two deletion mutants of StpkaC2 of Setosphaeria turcica,and transcriptome data showed that the encoding gene of StRab4 was significantly up-regulated in these mutants.Therefore,a StRab4 gene silencing vector was constructed in this study,and three StRab4 gene silencing mutants were obtained by PEG-mediated protoplast transformation method,in order to clarify the function of StRab4 gene in the infection of Setosphaeria turcica.The main research results are as follows:1.The gene with Protein ID 162018 was searched in the database of Setosphaeria turcica published by JGI,and it was named as StRab4 according to its conserved domain.The total length of the gene is 1455 bp,containing four exons and three introns.The CDS sequence length is 1002 bp and encods 333 amino acids.The subcellular location of the gene is on the Golgi membrane.The amino acid sequences of Rab were analyzed by Protein BLAST of NCBI and SMART domains,and it was found that there are a total of 10 gene coding products in the genome of Setosphaeria turcica that have Rab conserved domains,and the amino acid sequence homology between them ranged from 28.57%to 55.17%.2.The StRab4 gene silencing vector was transformed into the wild-type of Setosphaeria turcica by PEG-mediated protoplast transformation.After screening and verification,three mutant strains were obtained,named StRab4i-1,StRab4i-2 and StRab4i-4,respectively.The expression level of StRab4 gene in WT and three strains of StRab4i mutant was analyzed by qPCR assay,and the results showed that the expression level of StRab4 gene in the mutants was significantly lower than that in WT,which was 0.58,0.47 and 0.42 times of WT,respectively.3.Compared with the wild-type strain,the colony color of the three StRab4i mutants became lighter,the growth rate slowed down,the amount of conidium production was significantly reduced,and some of the conidia had abnormal morphology,indicating that StRab4 gene regulates the hyphae growth and conidial development of Setosphaeria turcica.4.The cell wall integrity of WT and StRab4i mutants were analyzed by using PDA containing 100 μg/mL Congo Red,20 μg/mL CFW and 0.01%SDS.The results showed that Congo Red,CFW and SDS had a lower inhibitory rate on the mutants than WT.The surface hydrophobicity test showed that the wettability area of the three StRab4i mutants was larger than that of WT.These results indicated that the cell wall integrity of the mutant was decreased,which indicated that StRab4 gene affected the cell wall integrity of Setosphaeria turcica.5.The chitin staining of the hyphae of WT and StRab4i mutants showed that a large amount of chitin accumulated at the top of the hyphae of WT strain,while the chitin at the top of the mutant hyphae was not obvious.The chitin content of WT and StRab4i mutants was determined,and the results showed that the chitin content of the mutants was significantly lower than that of WT.The results indicated that the StRab4 gene could regulate the synthesis and transport of chitin in Setosphaeria turcica.6.The intracellular melanin of WT and StRab4i mutants was extracted,and the content of intracellular melanin in the mutants was significantly lower than that in WT.The extracellular melanin of WT and mutants were extracted under the condition of shaking and static culture,the results showed that under different culture conditions,the extracellular melanin content of WT changed little,but the extracellular melanin content of the mutants under shaking culture conditions was significantly higher than that of the cultures under static conditions,indicating that the melanin of the mutants was more easily secreted to the outside of the cells under shaking culture conditions.7.The conidial germination of WT and StRab4i mutants was induced on cellophane to detect the penetration ability of the mutants.The results showed that the formation rates of germ tubes,appressorium and penetration peg of mutant conidia were lower than those of WT.The maize leaves were infected with the same amount of WT and StRab4i mutants conidia.It was found that after infecting the maize leaves,the early infection courts formed of the mutants were less than that of WT.and the lesion area was also smaller than that of WT,which indicated that StRab4 gene affected the infection ability and pathogenesis of Setosphaeria turcica.8.The morphologically abnormal conidia in the StRab4i mutants were isolated from monospores and named StRab4i-S.The expression level of StRab4 gene in the StRab4i-S strain was analyzed by qPCR assay,and the results showed that the expression level of the gene in the StRab4i-S strain was significantly lower than that in WT,which was 0.29 times of WT.Compared with the WT strain,the subculture ability of StRab4i-S strains was greatly weakened.The spore morphological abnormality rate exceeded 2/3,and the size was close to 1/2 of WT,the germination and penetration ability of the spores was significantly reduced,the distance between hyphal septa was longer than that of WT,indicating that StRab4 gene has a regulatory effect on the morphogenesis of the conidia of Setosphaeria turcica. |
