Functional Analysis Of SRNAs And StTET8 In Regulating Potato Late Blight Resistance

Potato(Solanum tuberosum L.)is the fourth largest food crop in the world.China ranks first in the world in terms of potato planting area and total yield.Potato plays an important role in ensuring food supply and increasing farmers’ income.Late blight,caused by Phytophthora infestans(Mont.)de Bary,is the most serious disease of potato,causing serious damage to production.The current main method of preventing and controlling late blight is through chemical pesticides,however heavy application of chemical agents will increase production costs,seriously endanger the environment and human health,and is not conducive to the green and sustainable development of the potato industry.Therefore,investigating the immune regulation mechanism of potato late blight resistance,exploring effective disease control strategies,and creating genetic resources are of very important for potato late blight resistance breeding practice.sRNAs play an important role in regulating plant immune response.In the long-term arms race between plants and pathogens,sRNAs not only regulates plant targets,but also cross kingdom to pathogens to silence pathogenic virulence genes and weaken their pathogenicity.To explore the role of sRNAs in the immune response,this research used stem-loop q RT-PCR method to detect the expression level of sRNAs in potato at different P.infestans infection stages.Resistance function of sRNAs was confirmed through transient expressing in Nicotiana benthamiana and stable overexpressing in N.benthamiana and potato transgenic lines.Function identification of target genes were conducted by using the transient expression in N.benthamiana combined with P.infestans inoculation method.In addition,this study we generated a stacked expression vector for the cross-kingdom transportable sRNAs and the extracellular vesicle-related gene StTET8,aimed to enhance the potato late blight resistance by increasing the efficiency of sRNAs cross-kingdom transportation.The main results are as follows:1.miR394,miR6149-5p,novel-s RNA133,and novel-s RNA140 expression levels were down-regulated in potato leaves responds to P.infestans infection,indicationg those sRNAs may participate in regulating potato immune responses.2.Compared with the control,miR394 transient expression in the leaves of N.Benthamian,or stable overexpression in potato and N.benthamian transgenic lines significantly increased the disease lesion area,and decreased the ROS(reactive oxygen species)burst.The above results indicates that overexpression of miR394 inhibited ROS burst and weaken potato resistance to late blight.In the stable overexpression lines of miR394,the target genes St A/N-INVE and St LCR were downregulated.Co-expression of miR394 with St A/N-INVE-GFP,or GFP-St LCR decreased GFP fluorescence intensity in N.benthamian.The above results indicate that miR394 could degrade the target genes St A/N-INVE and St LCR.Transient overexpression of target genes St A/N-INVE and St LCR in N.benthamian enhance late blight resistance.The above results indicate that miR394 down regulates the target St A/N-INVE and St LCR to weaken potato late blight resistance.3.Transient expression of miR6149-5p in the N.benthamian did not affect late blight resistance.There is no significant difference in lesion diameter compared to the control.However,miR6149-5p stable overexpression in potato shown reduced the P.infestans lesion area compared with the control,indicating that miR6149-5p positively regulates late blight resistance in potato.4.novel-s RNA72,novel-s RNA121,novel-s RNA133 and novel-s RNA140 derived from potato were only predicted the target genes in P.infestans genome.Four sRNAs(4sRNAs)was stacked in one construct.Transient expression of 4sRNAs in N.benthamian,or stable overexpression in potato transgenic lines significantly decreased the disease lesion when inoculated with P.infestans,compared with the control The results indicate that 4sRNAs may target P.infestans genes to enhance potato resistance.The ROS burst in 4sRNAs stable overexpression potato lines were significantly higher than those in control,indicating that overexpressing 4sRNAs induced ROS burst and enhance potato late blight resistance.5.StTET8 is a component of extracellular vesicles that helps cross-kingdom sRNAs transportion.In this study we found that stable overexpression of StTET8 in N.benthamian or potato significantly increased late blight resistance.The function of StTET8-4sRNAs stacked vector was verified by transient overexpressing StTET8-4sRNAs in N.benthamian,the results showed that compared to the overexpression of StTET8 or 4sRNAs alone,StTET8-4sRNAs stacked vector significantly enhanced the resistance of N.benthamian to late blight.In summary,the results of this study indicated that sRNAs derived from potato involved in the regulation of late blight resistance,some of sRNAs positively regulated resistance,while the other negatively regulated resistance.Late blight resistance could be significantly improved by co-expression of the P.infestans-targeted sRNAs and StTET8 which is a component of extracellular vesicle complex helping sRNAs cross-kingdom transportion.This study provides examples and ideas for using sRNAs to regulate potato late blight resistance.