| Setosphaeria turcica, an important fungal pathogen, gives rise to a serious threat to corn production. Substantial yield losses of corn are caused by periodic epidemics of this plant disease. Lots of researchers indicate that Ras protein acts as a molecular switch that exists in active (GTP bound) state or inactive (GDP bound) state. It is the important organizer in cAMP, MAPK and Ca2+ signaling pathways. Through these signal transduction pathways, Ras protein can regulate the growth, development and pathogenesis of pathogenic fungi.In this study, we identified the Ras homology acquired in our lab using a conserved domain searched from NCBI. Bioinformatic analysis was conducted using sequence alignment in Ras proteins from S. turcica and other fungi. The results revealed that StRas protein was closely related to Ras2 in other fungi. It consists of four GTP/GDP domains, an effector binding domain and a CAAX motif as Ras2. The most striking difference is that Ras2 has approximately 20 amino acid insertion compared with Ras1. The insertion could be found in most members of the Ras2 homologs but not in Ras1, which further proved that the cloned gene could be named as StRas2.With an aim to define the relationship between StRas2 gene and S. turcica, RNAi (RNA interference) were applied in the present study. The silencing vector pSilent-1- StRas2-F-R was constructed and transformed into protoplasts of wild-type strain by PEG4000. The transformants were screened by the medium with hygromycin B added. Furthermore, six StRas2-silencing transformants, which were named as R1, R2, R3, R4, R5 and R6 respectively, were identified as positive by amplifying hygromycin B phosphotransferase gene using its specific primers. The expression level of StRas2 was analyzed by semi-quantitative RT-PCR. The results showed that the expression of StRas2 mRNA in these transformants was weakened in different levels. Phenotypic analysis of these transformants showed dense and velvet aerobic mycelia and shallow colonial color. The hyphae morphology of wild type strains and transformants were observed respectively by microscopy. The hyphae of R1 and R2 transformants with StRas2-silenced higher efficiency displayed a stunted phenotype, with frequent diversion of the axis of polarity, and their hyphal diameter was more than twice as long as that of wild type strains. From the analysis of the growth rate, we found that the growth rate of transformants were decreased, with the same degrees of the declined StRas2 silencing efficiency. Statistical analysis of the conidial production showed that the conidial yield was reduced when StRas2 weakly expressed. R1, R2, R3 and R4 could not produce conidia. R5 and R6 could produce conidia with similar conidial morphology of wild type strains, however, the conidial yield was significantly decreased. In the penetration assay, appressoria formed by 01-23, R3, R4, R5 and R6 could produce invasive hyphae on the surface of artificial cellophane. With the same inoculation time, R1 and R2 could not form the appressorium. R3, R4 displayed a severe delay when forming appressoria and producing invasive hyphae that can penetrate the artificial cellophane. The typical diamond-shaped lesions caused by transformants were smaller than those caused by wild type strains. R1 and R2 were unable to cause disease symptoms. We also found that the biological activity of HT-toxin from all transformants and wild type strains showed no difference. All these results indicate that StRas2 is involved in the growth, development, conidial generation and appresoria formation of Setosphaeria turcica and is not related to the biosynthesis of HT-toxin. |
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