Effects Of Small RNAs From Four Genes Of Fuasrium Oxysporum F.sp. Cubense Tropical Race 4 On Its Early Development Stages In Vitro

Fusarium wilt of banana caused by Fuasrium oxysporum f. sp. cubense tropical race 4(Foc TR4), threatens vital banana-growing regions worldwide, which is a world problem that needs to be solved. Some researches showed that host plant-induced gene silencing(HIGS) technology is one of the effective methods to develope new germplasms with broad-spectrum and durable resistance against insects,nematodes, bacteria and fungi. Based on early researches on proteomic analysis of conidia germination in Foc TR4 and genomic analysis of 24 vegetative compatibility types,we found that ERG6, ERG11, ERG13 and ERG25 involved in ergosterol biosynthesis pathway are the vital and key proteins in Foc TR4 early growth, which could allow us to develop novel transgenic banana leading to high protection from Fusarium wilt and to explore more effective antifungal drugs against either individual or multiple target proteins of Foc TR4. In this study we constructed prokaryotic expression vectors containing one of above four genes with the hairpin structure(hp RNA), which can express integrated ds RNA in E.coli HT115 strain(RNase III deletion). We explored suppression of Foc TR4 and Foc Race1's early growth and their related molecular mechanism by feeding ds RNA of the four genes in vitro. The main results are as follows:1. Construction of prokaryotic expression vectors and RNAi vectors Four optimized ds RNAs fragments of ERG6, ERG11, ERG13 and ERG25 from Foc TR4 were expressed through prokaryotic expression vectors and plant expression vectors. The prokaryotic expression vectors were transformed into E.coli HT115 to express integrated ds RNAs and their expression levels were detected by Real-timePCR. Transgenic bananas containing Fo ERG6 and Fo ERG11 interference fragments were obtained by HIGS technology.2. The ds RNAs of four target genes can significantly suppress Foc TR4 and Foc race1's early development in vitro The ds RNAs of above four genes' have significantly inhibited the effects on Foc TR4 and Foc race1's early development, in which the fungi with 1×105 conidia/ml were treated with 500ug/ul ds RNA and evaluated for growth inhibition at 12, 24 and48 hours. The suppression effect of Fo ERG11 and Fo ERG25's ds RNAs was better than that of Fo ERG6 and Fo ERG13. It is also suggested that the four target genes' sequence from Foc race1 are highly conserved with Foc TR4.3. The gene expression profiles of the above four genes and other genes involved in ergosterol and cholesterol biosynthesis pathways of Foc TR4 When Foc TR4 was treated by the aboved four genes' ds RNAs for 12 h, the four corresponding genes and others' genes(ERG3A?ERG3B?ERG4?ERG6A?ERG6B?ERG11A ? ERG11 B ? ERG11 C ? ERG13 ? ERG11 A ? ERG11 B ? ERG11 C and ERG25)involved in ergosterol biosynthesis pathway were significantly down-regulated, but the genes involved in cholesterol biosynthesis pathway(TGL?ARE1A and ARE1B) were significantly up-regulated as analyzed by quantitative PCR.Feeding four target genes of ds RNA, can not only effectively inhibit the expression of their corresponding genes, but also suppressed the other genes involved in the ergosterol and cholesterol biosynthesis pathway This may also be the molecular mechanism underlying the inhibition of Foc TR4 early development.4. RNA-Seq analysis of expression of small RNAs in the transgenic banana Transgenic banana strains with Fo ERG6, Fo ERG11 and wild type were grown in nutrient solution containing spore concentration of 1×105 conidia/ml, after 11 days,the leaf samples from the transgenic banana and wild type were sent to sequencing.The results found that after the treatment of pathogens, the proportion of small RNA expression in the four strains(RNAi- ERG6-8, RNAi- ERG6- 36, RNAi- ERG11-43, RNAi- ERG11 – 52) were 1.96 %, 2.02 %, 5.47 %, 6.20 %, respectively and alsosome new mi RNA were found. In addition, the target gene of small RNAs were also predicted.Our current study demonstrates a prospective way for controlling banana wilt using ds RNA through the HIGS approach. The study strongly support that HIGS is a highly efficient strategy for controlling the growth and development of Foc TR4;Fusarium wilt can absorb the external feeding of ds RNA and trigger RNAi silencing mechanism in vivo; The transgenic banana strains show strong resistance to Foc TR4.It is suggested that that the host plant induced gene silencing(HIGS) is one of the effective methods to cultivate new germplasm with broad spectrum and durable resistance.