| Nomuraea rileyi, an environmentally friendly entomogenous fungus, infects avariety of noctuids. Due to its important role in pest control, it has an immense potentialto develop to be an insecticide. As a pesticide, the conidia of entomogenous fungi arethe main effective components of pesticides. However, the sporulation of N. rileyirequires some special growth conditions, such as a specific carbon source (maltose) andlight stimulation, which limit its mass production and commercialization. Therefore, weresearched it with a different approach, and successfully induced microsclerotium (MS)of N. rileyi formation in liquid amended medium (AM). And the MS can be an infectstructure to replace the traditional infect structure of conidia.MS is a special reproductive structure and has advantages of strong environmentresistance, resistance to high temperature and storage resistance. But the mechanismunderlying MS development in entomogenous fungi is obscure. In order to explore themolecular developmental mechanism of MS, large-scale production of MS to controlinsects as well as to lay a foundation for developing efficient cutworm biological agents,our group had carried out some researches on the comparative transcriptome analysis ofMS development in N. rileyi. According to these results, our studies were conductedfrom the following two aspects in the present study: one was the relationship betweenoxidative stress and differentiation of MS; the other was that an NADH: flavinoxidoreductase/NADH oxidase (Nox) gene was cloned and RNA interference (RNAi)technology was used to analyze its function. The main results were as follows:①Exogenous oxidative stress producing agents (H2O2, menadione) and anantioxidant (ascorbic acid) were added to AM for N. rileyi growth to explore the effectsof reactive oxygen species (ROS) on MS differentiation. The results showed that thebiomass and MS yields of N. rileyi were significantly increased following the treatmentswith107and105M H2O2. Addition of1μM menadione to AM also significantlyincreased the MS yields. However, ascorbic acid was disadvantageous to MS formation.It resulted in a dose-dependent decrease in MS formation under low-oxidative stress.Furthermore, there was no puce pigment in the AM, but only hyphae or mycelial pelletsin AM when treated with0.03g ascorbic acid l1. This suggested that a certainconcentration of ROS could promote MS differentiation.②The intracellular H2O2level exhibited that the H2O2level peaked in the MI stage, which was about3-fold higher than that observed in the UD stage. These resultsindicated H2O2played an important biological role in MS development, namely the highlevel of ROS involved in the MS differentiation.③Based on the transcriptome library, an NADH: flavinoxidore ductase/NADHoxidase gene (Nox) was cloned from N. rileyi with RACE technology (GenBankAccession No. KF425266). The1663bp full-length cDNA contains an open readingframe of1233bp coding410amino acids. The Nox genomic sequence of1788bpconsists of three exons interrupted by two introns, corresponding to the cDNA sequence.Analysis of the Nox protein reveals that the predicted molecular mass as44.81kDa,isoelectric point as7.12and there is no signal peptide as well as transmembranestructure. Furthermore, the Nox protein locates in the mitochondria, which contains anold yellow enzyme (OYE)-related FMN binding domain (15-356amino acids). Themultiple sequences alignment and phylogenetic tree indicated that the Nox protein of N.rileyi had the highest homology (82%) as well as the closest genetic relationship to thatof Metarhizium spp.④The transcription level of N. rileyi Nox during MS development was analyzedby RT-qPCR. The results demonstrated that the mRNA level of Nox expressed in alldevelopmental stages, but dramatically increased during MI stage, exhibiting5.6-foldhigher than that observed during UD stage, but then it decreased. The expression levelof Nox was co-related to the intracellular H2O2concentration during MS differentiation.⑤An enzyme inhibitor and RNA interference technology were used to analyze thefunction of Nox gene, these showed that the Nox gene was involved in the developmentof MS. The research of inhibitor found that40μM and100μM rotenone couldsignificantly inhibit the formation of MS. Furthermore, the Nox mRNA expressionapproximately decreased by50%following the addition of100μM rotenone.⑥The efficiency of RNAi in the transformants was analyzed by RT-qPCR. Itindicated that500nM siRNA appeared to be effective for Nox silencing, where the Noxtranscription showed a significant reduction of57%. And there were obvious differencesin morphological features after gene silencing. Following the treatment with500nMsiRNA, the fermentation broth exhibited that the dark purple pigment disappeared,viscosity declined and the size of MS was larger than that noted in the controls.Furthermore, the Nox-RNAi strain could hardly form MS, in which the MS yieldssharply decreased by98.5%. The results showed that the Nox involved in regulating theMS formation. ⑦The Nox gene related to intracellular H2O2production. The intracellular H2O2level of500nM siRNA Nox-RNAi strain was dropped by38%. In summary, wepresumed the Nox gene regulation of MS differentiation mechanism: the Nox maycontrol MS differentiation by changing its transcription to regulate the intracellularH2O2accumulation.⑧The Nox gene also influenced the virulence of MS in N. rileyi. Analysis of thevirulence of the MS produced by Nox-RNAi strain for cabbage caterpillar larvae, itindicated that the average LT50values for RNAi strain was significantly longer than thatthat of wild-type strain. It showed that silencing of the Nox gene, the virulence of MSdecreased. |
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