Functional Identification Of StMLP1 Regulating Potato Bacterial Wilt Resistance

Bacterial wilt caused by Ralstonia solanacearum is a soil-borne vascular disease threatening the potato industry.The bacteria can enter the xylem of the plant via the natural orifice and wound,and be transported upward through water and adsorbed to the inner wall of the vessel.R.solanacearum can not only grow into aggregates to block vessels,but also release effectors extracellular and intercellular to destroy plant immunity.The plants wilt and died eventually due to the disorder of the immune system and water shortage.The complexity of R.solanacearum and the high heterozygosity of potato make it difficult for potato breeding to resist bacterial wilt.The most green and effective breeding method is to make full use of potato germplasm resources to screen and create new resistant materials.However,at present,it is difficult to find resistant resources to fight bacterial wilt,let alone clone resistance genes.Therefore,researchers focus on finding resistance related genes and exploring the mechanism of plant-pathogen interaction.Kunitz Trypsin Inhibitors(KTIs)are a large family of protease inhibitors that have been well studied,and can play a certain role in plant resistance.A miraculin-like protein(MLP)previously screened by our group.It belongs to KTIs with a complete Kunitz domain.But its function and mechanism in the interaction between potato and R.solanacearum remain unclear.This study systematically analyzed the function of this MLP and provided theoretical basis for the resistance mechanism of potato bacterial wilt disease.The results as follows:1.StMLP1 was specifically expressed in the vascular bundle of potato after induction by Ralstonia solanacearum.Sequence analysis showed that the MLP had a high homology with Lemir in tomato.Among the 7 KTIs of Arabidopsis,it had a higher homology with At KTI1,so it was named StMLP1 according to the naming sequence rule in Arabidopsis.Analysis of the gene expression pattern of StMLP1 in potatoes found that the gene was not expressed in normal conditions.After bacteria inoculation,the gene expression level in root and stem has increased gradually as the deeper bacterial infection,indicating that StMLP1 could respond to bacterial infection.The promoter sequence analysis showed that except some important elements related to biological stress,there was also a conserved sequence of vascular specific binding expression.Transient expression of OE-Pro MLP::GUS in tobacco leaves was mainly expressed in leaf veins,which indicated that StMLP1 was specifically expressed in vascular bundles.OE-Pro MLP::GUS was steadily expressed in potato E3,which was mainly expressed in stem and root,further indicating that the promoter of StMLP1 has the function of specific expression in the vascular bundle.2.StMLP1 was a protease inhibitor that negatively regulated potato bacterial wilt,and the signal peptide played an important role in its correct localization in cells.The StMLP1 fusion protein after removing the signal peptide was purified by prokaryotic expression,and the trypsin inhibitor activity was detected.The results showed that StMLP1 could inhibit the trypsin activity.In the experiment of subcellular localization,after the signal peptide was removed from StMLP1,a large number of proteins gathered in the nucleus;while the signal peptide was retained,the protein was mainly near the cell membrane,indicating that the signal peptide played an important role in helping StMLP1 performing function at the correct position.For further functional verification,we obtained StMLP1 overexpression and interference stable transgenic lines of potato.The identification results of bacterial wilt resistance showed that overexpression of StMLP1 reduced the resistance of plants to bacterial wilt,while interference lines significantly increased the resistance of plants to bacterial wilt.These results indicated that StMLP1 negatively regulated potato resistance to bacterial wilt.3.StMLP1 was involved in regulating the resistance pathway of potato bacterial wilt.RNA-Seq was performed on root samples of StMLP1 overexpressed lines and E3.Some potatoes differentially expressed genes were obtained.Multiple pathways related to plant immunity were enriched through GO and KEGG analysis.Compared with E3,these genes were significantly up-regulated in overexpressed lines,indicating that StMLP1 may be involved in these pathways to regulate potato resistance to bacterial wilt.However,the interaction mechanism remains to be further verified.