The Predictive Analysis Of The Secreted Proteins And The Protein Filtering About Inducing Resistance In Saccharothrix Yanglingensis Hhs.015 Genome

Saccharothrix yanglingensis strain Hhs.015,being a biocontrol agent,isolated from cucumber root,has shown positive effects on the control of tomato leaf mold and apple Valsa canker disease,of which the biological control mechanism is unknown.Pervious study indicates a potential role of Hhs.015 on inducing plant resistance.The secreted proteins may be responsible for inducing plant resistance.In this study,we predicted the potential secreted proteins using a computer pipeline based on the genome data,evaluated the roles of several putative proteins on inducing plant resistance,and detected the change of defense-related enzymes in apple leaves inoculated by one obtained elicitor.The main results are as follows:(1)Three hundred putative secreted proteins,of which 56 percent with 100 – 400 amino acids in length,were predicted using a computer pipeline comprised of SingnalP 4.1,TMHMM,PrositeScan and big-PIpredictor programs based on Hhs.015 genome data.Subsequently,the obtained genes were characterized through CDD search and blast2 go annotation.The signal peptides mainly have 23– 27 amino acids in length,with a classic AXA cleavage site.And 158 proteins were annotated,mainly as enzymes such as serine proteases,peptide enzymes,etc.(2)Twenty-nine putative protein genes,selected from 142 putative proteins with unknown function,were heterogeneously expressed using a pET28 a system in Escherichia coli BL21.First,the expression system was optimized.Six proteins,named as 5620 g,1132g,194 g,2349g,3176 g and 3638 g,were successfully expressed after an induction with 0.1 Mm IPTG for 14-18 hours under 16-18oC,and purified with His-Trap Ni–Sepharose.(3)Bio-assays on Nicotiana benthamiana and apple leaves were performed for evaluating roles of the six expressed proteins on inducing plant resistance.All of them,injected into Nicotiana benthamiana leaves via a bunted syringe,could not induce any visible symptoms.Five of them,except 5620 g,could not induce resistance on apple leaves against Valsa mali.Remarkably,apple leaves soaked for 10 minutes in 5620 g protein extracts showed significantly improved resistance against Valsa mali.(4)The accumulation of defense-related enzymes could be induced by 5620 g protein extracts in apple leaves.Three enzymes,peroxidase(POD),phenylalanine ammonia lyase(PAL)and polyphenol oxidase(PPO),were detected at 1,2,3,4,5,6 days post protein-soaked treatment of apple leaves.POD activities highest value at the third day,which was 58.2% higher than that of control.PAL activities increased 113.8% at the third day in the protein-treated leaves compared to the control.PPO activities significantly increased at the fourth day in the protein-treated leaves as well.In summary,this study screened an elicitor 5620 g showing extremely good effects on inducing apple resistance,which can be used to unveil the mechanism underlying induced resistance and can be developed further for agricultural application.Moreover,this study provides a framework for identification of elicitors in bio-control agents based on genome data.