Structural And Functional Analysis Of StPBS2Gene Ecoding A MAPK Kinase In HOG-MAPK Cascade Pathway Of Setosphaeria Turcica

Setosphaeria turcica (anamorph Exserohilum turcicum) is the causative agent of thenorthern leaf blight of corn, which gives rise to a serious threat to corn production andreduces the yield of corn up to50%in the disease epidemic years. However, controlefficiency of chemical pesticides on northern leaf blight of corn is not ideal. Therefore, thesearch for new control methods and preventive measures have become imperative. Inrecent years, exploring the development and pathogenic molecular mechanisms of plantpathogenic fungi and seeking more effective prevention and control measures have becomethe most popular research topics in the field of plant pathology and plant genetics andbreeding. In previous study, a MAPKK gene named StPBS2, which belongs toHOG-MAPK signal transduction pathway of S.turcica, had been cloned and two StPBS2RNAi transformants had been preliminarily identified using hygromycin B resistance andRT-PCR technique. Based on the primary research, we analyzed the structuralcharacteristics of StPBS2gene using bioinformatics technology, and determined the RNAitransformants and the expression levels of StPBS2gene employing Real-time PCRtechnology in this study. The main results were listed as follows.1. The precise location of StPBS in the genome, physical and chemical characteristics,space structure, conserved domains of StPbs2were identified by searching the genomedatabase of S. turcica published on JGI website (http://genome.jgi-psf.org/). Homologousanalysis showed that StPBS2with high homology to Botrytis cinerea Bc-PBS2genebelonged to MAPKK-like gene in HOG-MAPK signal transduction pathway.2. Based on the analysis of Real-time PCR technique, two RNAi transformants (R3,R14) were determined and the expression levels of StPBS2in the transformants R3, R14were62%,38%, respectively.3. The result showed that the colony growth rate of the transformants decreasedsignificantly, the number of the conidium reduced, and the mycelial cells swelledcompared with the wild-type strain (WT). Moreover, the variation trend was positivelycorrelated with that of StPBS2gene silencing level, indicating that StPBS2was involved inthe regulation of mycelium development and conidium formation. 4. It was found that the cell wall turned thicker as the gene silenced in differenttransformants by observing the cell wall structure of StPBS2RNAi transformants.Furthermore, the sensitivity to cell wall synthesis interfering substances was detected.The results showed that the transformants were more sensitive to the cell wallsynthesis interfering substances compared to the WT, and the transformant R14, withhighest StPBS2silence level, appeared strongest sensitivity. These results indicated thatStPBS2was involved in regulating cell wall development of S. turcica.5. Under hypertonic stress, the colony morphology of the transformants changedsignificantly, the growth rate of them lowered obviously, and the trend displayed a positivecorrelation with the silencing efficiency of StPBS2, indicating that the gene was involvedin controlling hypertonic stress response of the pathogen.6. Under oxidative stress, the colony growth rate of the transformants reducedsignificantly compared to WT, and the trend displayed a positive correlation with thesilencing efficiency of StPBS2, indicating that StPBS2was involved in the positiveregulation to the oxidative stress response.7. Based on the fungicide sensitivity test, it was found that the resistance to fungicidesof the transformants distinctly increased as the gene silencing efficiency reduced. Furtherdetermination of the concentration for50%of maximal effect (EC50) of WT and thetransformant R14with the strongest silencing efficiency, the result showed that the EC50ofR14was100times higher than that of WT. These results showed that StPbs2was likely tobe the action site of fungicides including fludioxonil, iprodione and procymidone.8. By analyzing the red components of secondary metabolites of the RNAitransformants and WT using high performance liquid chromatography (HPLC)technonlogy, we found that WT and the transformants produced similar secondarymetabolites. Further analysis showed that the carbon source utilization levels of thetransformants and WT were positive correlation with the gene expression level in differentgene silence transformants. The results showed that the StPBS2gene might be involved inthe regulation of the produce of red secondary metabolites, and HOG-MAPK pathwaymight be involved in glucose metabolism.