| As a kind of environmental-friendly fungal pesticides, entomogenous fungi willnot cause environmental pollution, residual and resistance in control of insect pests.Along with the increase of environmental awareness, the research ofenvironment-friendly pesticides is widely strengthened. As an importantentomopathogenic fungus, Nomuraea rileyi is capable of infecting several lepidopterouspests, causing insect disease in natural environment. As an important bio-control agent,the effective component of N. rileyi is conidia, which require fastidious growthconditions along with stimulatory light, thus, limiting its mass production andcommercialization. Our laboratory successfully accomplished N. rileyi microsclerotium(MS) production in liquid amended medium (AM), which exhibited insecticidal efficacyon Spodoptera litura, and confirmed that MS can be used as an alternative active agentto conidia in pest bio-control. Subsequently, through transcription analysis, we foundthat MS differentiation associated with oxidative stress and polarized growth of hyphae.In the present study, we cloned the racA and cdc42genes that were upregulated duringMS differentiation and characterized their involvement in MS development andpolarized growth. The results of main research are as follow:①Cloning and identification of the target genes: The full-length cDNA sequencesof the N. rileyi racA and cdc42genes were obtained by SMART RACE RT-PCR. Theputative coding sequences of racA and cdc42were657and555bp, encoding218and184amino acid residues, respectively.②Bioinformatics analysis of the target genes: Protein sequence analysis foundthat both RacA and Cdc42harbor the typical five GTP/GDP-binding or hydrolysismotifs (G1–G5) and two molecular switches characteristic of the Rho-family smallGTPase; The phylogenetic tree analysis demonstrated that the RacA or Cdc42proteinsof N. rileyi are highly homologous to those of Metarhizium acridum.③The dynamic process of MS development: We observed the dynamic process ofMS formation with microscope and found that it was composed of winding polarhyphae and was accompanied by the dimorphic switch of hyphae (yeast-like cells andmycelium); in the period of MS mature, there accompanied by secondary mycelialgrowth in the bottles.④Transcription levels analysis of the target genes: The transcription levels of racA, cdc42and NOX complex were investigated by real-time quantitative PCR (qPCR)at different stages of MS development and showed that the transcription levels of cdc42,racA and NOX complex were highest in the SI and hyphal period, whereas that of racAand cdc42were highest in the stage of dimorphic switch.⑤Functional verification of racA and cdc42: To further assess the effect of racAand cdc42on MS development and fungal growth, we adopted the method of RNAi invitro with egfp which was used as a negative control. It was found that theRNAi-silenced strains, in particular, the racA&cdc42RM, exhibited severe reduction inthe extension rate and smaller colonies, when compared with the controls. Thedimorphic switch was delayed by approximately2and3days in the racARM andcdc42RM, respectively. Furthermore, when compared with controls, the conidial yieldof the racARM and cdc42RM was reduced to approximately47.4%and35.0%,respectively; on the other hand, the racA&cdc42RM exhibited no dimorphic switch (inthe yeast-like cells period) and hardly produced any conidia. When compared with theconidia of the controls, those of racARM were bigger and more rounded; those ofracA&cdc42RM were the biggest and not of uniform size, and those of cdc42RM werenot significantly different. The MS sizes of racARM were bigger than those of thecontrols, but smaller than those of cdc42RM. Furthermore, the MS yield of racARMand cdc42RM were reduced to approximately3–4%of that of the controls. In addition,silencing of both racA and cdc42limited the formation of MS (reduced to3‰), whosesize was different from that noted in the controls. When compared with the hyphae ofthe controls, those of the racARM were mostly thicker with increased apical branching,whereas those of the cdc42RM did not exhibit observable difference, except for anincrease in the number of branches. Furthermore, the hyphae of racA&cdc42RM weregenerally the thickest and had more branches than those of the controls.⑥ROS detection: ROS detection with NBT revealed that the levels of ROS inracARM and cdc42RM were substantially decreased, and in the racA&cdc42RM, ROSwas hardly detected.⑦Transcription levels analysis of the target genes after RNAi: The expressionlevels of noxA and noxR in three RNAi-slienced strains were reduced.⑧The virulence detection: The virulence of the all strains were tested byinoculation of the strains onto the cuticle of third-instar cabbage caterpillar larvae andfound mortality was reduced, the lethal time values for50%mortality (LT50) wasextended in the RNAi-scilenced strains. ⑨The relationship between the genes: The qPCR analysis demonstrated that thetranscriptional level of cdc42was higher in racARM, and that there was a smalldecrease in racA expression in cdc42RM. Furthermore, although the transcriptionallevels of noxA and noxR were decreased in the RNAi-silenced strains, the levels notedin the cdc42RM were lower than those observed in the racARM, and they were lowestin the racA&cdc42RM. Subsequently, the expression levels of cdc24were higher thanthe controls in racARM and cdc42RM.In conclusion, racA and cdc42genes were cloned from the entomopathogenicfungus, N. rileyi, and they appear to share some essential functions in N. rileyi,including hyphal growth, conidiation, MS formation, ROS generation and virulence. Yet,RacA appears to have more pivotal role in the polar growth of N. rileyi. Furthermore,we also demonstrated that during MS formation, racA and cdc42could influence ROSproduction by regulating the activity of the NOX complex. |
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