| Sclerotinia stem rot(SSR)is one of the most important fungal diseases caused by Sclerotinia sclerotiorum,which seriously threatens the yield and quality of rapeseed.S.sclerotiorum infected host plants through secreting cell wall degraded enzymes(CWDEs),polygalacturonase(PG),and pathogen associated molecular pattern(PAMPs).In order to resist the infection of pathogens,the plant evolved a class of polygalacturonase inhibition proteins(PGIPs)to inhibit the activity of PGs.In our studies,Os PGIP2 from rice(Oryza sativa)was overexpressed in Brassica napus lines 7-5(lightly resistance),P61-5(highly susceptible)and T45(highly susceptible),and 16 transgenic lines with significantly improved S.sclerotiorum resistance were screened by resistance identification and molecular identification in T1 generation.Then the T2,T3 and T4 transgenic lines were successively identified by molecular identification,inoculated with S.sclerotiorum and investigated seed quality and 1000-seed weight,and the results showed that S.sclerotiorum resistance was significantly improved in the transgenic rapeseed lines.Moreover,we used RNA-seq technique to analyze the differential expression genes between transgenic and non-transgenic lines after inoculation,and proposed the infection-defense network of Os PGIP2-mediated S.sclerotiorum resistance in the transgenic lines.The main results are as follows: 1.Identification of transgene copy number and expression analysis of Os PGIP2 in transgenic linesThe Southern blot results showed that the transgenic lines 7-5B,7-5C,7-5D,7-5G,7-5H,7-5J,T45B#1,T45B#2 and T45 C lines had one copy,7-5A,P61-5A,P61-5B#1 and P61-5B#2 had double copy,and 7-5M had five copies of the transgene.The q PCR results showed that there was significant difference in the expression of Os PGIP2 from the different transgenic events;there was no significant difference in the expression level of Os PGIP2 in the same transgenic event;the expression level of Os PGIP2 gene was the lowest in 7-5M with five copies of the transgene.Moreover,evaluation of S.sclerotiorum resistance in the T2 and T3 generations showed that except for the 7-5M transgenic line,other transgenic lines had a significant increase in SSR resistance compared with non-transgenic lines.These results indicated that the Os PGIP2 gene had been successfully integrated into the genome of B.napus,and the low-copy insertion event of the Os PGIP2 gene could increase the resistance of B.napus,and multiple copies of the transgenic event may lead to gene silencing.2.Evaluation of S.sclerotiorum resistance,1000-seed weight and seed quality in the transgenic Os PGIP2 linesThe area of the lesions on the detached leaves of T4 transgenic lines was significantly smaller than that of non-transgenic lines at 72 hpi;all the transgenic lines had a good seedling resistance in the survival test;simulation of extreme disease stress tests showed that the S.sclerotiorum disease incidence of the transgenic lines was lower than that of non-transgenic families at the adult stage;the size of the lesions on the stem was significantly smaller than that on non-transgenic lines in both the field and green house.When the plants were infected with S.sclerotiorum,the content of erucic acid and protein in the seeds of transgenic plants were not significantly different from the controls,but 1000-seed weight and oil content were significantly higher than those in non-transgenic lines.These results demonstrated that the transformed Os PGIP2 gene could improve the seedling and adult S.sclerotiorum resistance of B.napus lines 7-5,T45 and P61-5,and could guarantee the seed quality traits and 1000-seed weight less affected by S.sclerotiorum.3.Analysis of the Os PGIP2-mediated defense pathways to S.sclerotiorumThe RNA-seq results showed that the pathway of Os PGIP2-mediated defense response was mainly dependent on the regulation of RLKs(receptor-like kinases),Ca2+ and ion transporting,redox reaction and elevated H2O2 related genes,JA/ET and auxin,transcription factors,pathogen related defense response,cell wall reconstruction,CYP/GSH/ Histone,carbon source metabolism and transport,and other related plant innate immune response pathway.DAB staining and q PCR demonstrated that the accumulation of H2O2 and the expression levels of peroxidase gene and peroxide scavenger gene were higher than that in non-transgenic lines.By detecting cell wall composition profile in the stems of the transgenic lines and their parents,we found that the contents of arabinose,xylose,cellulose,mannose and lignin gradually increased following the development of florescence.In the final flowering period,the content of cellulose,hemicellulose and mannose of Os PGIP2 transgenic lines were significantly higher than that of non-transgenic,indicating that overexpression of Os PGIP2 may affect the cell wall carbohydrate-related pathways to increase SSR in the transgenic rapeseeds.4.Analysis the interaction between Ss PGs and Os PGIP2We cultured S.sclerotiorum on PDA plate added with leaf extraction,and found that the growth of S.sclerotiorum in the medium with Os PGIP2 transgenic leaf extract was significantly delayed,and the arrangement of mycelium was tight,while the growth of S.sclerotiorum in the medium with the leaf extact of no transgenic lines was rapidly and the mycelium was loosely arranged.q PCR analysis of the S.sclerotiorum PGs showed that the expression of Ss PGs was low expression in the stem of transgenic lines than that of the non-transgenic lines.Comparing the affinity between Os PGIP2 and S.sclerotiorum PGs,the results showed that the affinity between Os PGIP2-Ss PG3 and Os PGIP2-Ss PG6 was higher than between Os PGIP2-Ss PG1 and Os PGIP2-Ss PG5.At the same time,the splite luciferase(LUC)interaction experiment further confirmed that Os PGIP2 could interact with Ss PG3 and Ss PG6,but without interacting with Ss PG1 and Ss PG5.In summary,overexpression of Os PGIP2 can significantly increase seedling and adult S.sclerotiorum resistance in Brassica napus without impairing 1000-seed weight and seed quality traits.Leaf extracts of B.napus plants overexpressing Os PGIP2 showed enhanced S.sclerotiorum resistance by delaying pathogen infection.The constitutive expression of Os PGIP2 in rapeseed plants increased the production of reactive oxygen species,increased the cell wall cellulose,hemicellulose and mannitol content,interacted with S.sclerotiorum polygalacturonases(Ss PG3 and Ss PG6),and effected on the expression of genes related to innate immune response.The results demonstrated that Os PGIP2 plays a major role in rapeseed defense mechanisms. |
PDF Full Text Request