Silencing Effects Of Map-1 And Cg-1 Genes On Virulence Of Meloidogyne Incognita Using RNAi Technique

Root-knot nematode(RKN,Meloidogyne spp.)is one of the most important group of plant parasitic nematodes,which causes tremendous damage to agricultural crops.RKNs can secrete effectors to plants and induce them having the effector-triggered immunity(ETI),which is similar to fungus and bacteria.Until now,a few effectors(avirulence genes)in RKNs were discovered,including the map-1 gene and Cg-1 gene which are the avirulence-related genes that corresponds to the resistant gene Mi in tomato.The map-1 gene is originated from Meloidogyne incognita and Cg-1 gene is from M.javanica.In this study,in vitro RNAi using dsRNA soaking and in vivo RNAi mediated by Tobacco rattle virus(TRV)were used to study the silencing effects of map-1 and Cg-1 genes on virulence and parasitism of avirulent M.incognita population.The results are as following:1.The J2s of avirulent M.incognita population after soaking in map-1 dsRNA and Cg-1 dsRNA solution infected the resistant tomatoes and completed their life cycle successfullyThe second-stage juveniles(J2s)were soaked in map-1 dsRNA and Cg-1 dsRNA solutions respectively.Six hours later,the transcription levels of the target genes in J2s were detected by RT-qPCR.The results showed that the transcription levels of map-1 and Cg-1 genes were significantly reduced when compared with the J2s soaked in gfp dsRNA used as a negative control.Meanwhile,the results of phenotype detection showed that J2s after soaking in gfp dsRNA solution produced few galls on resistant tomatoes without producing egg masses.However,the J2s after respectively soaking in map-1 dsRNA and Cg-1 dsRNA solutions,successfully infected the roots of resistant tomatoes and developed into normal females which can induce the root galls and produce the egg masses.These phenotypes demonstrated that dsRNA soaking of target genes resulted in a certain degree effects of avirulence/virulence of M.incognita,which enhances successful infection and reproduction on Mi-1 gene tomatoes.The results indicated that the avirulence related genes map-1 and Cg-1 may play an important role in the incompatible interaction between M.incognita and resistant tomato.2.The reproduction ability of J2s of avirulent M.incognita population was enhancedon resistant tomatoes with Agro-infection of TRV::map-1and TRV::Cg-1In vivo RNAi mediated by TRV was used to study the silencing effects of map-1 and Cg-1 genes on avirulence/virulence of M.incognita.The transcription level of cp gene in TRV was detected in roots of resistant tomatoes at 7 and 14 days after Agro-infection by RT-RCR.The J2s of avirulent population MIJS3-AV inoculated on resistant tomatoes Agro-infected with TRV::gfp only produced very rare galls without egg masses.Meanwhile,J2s produced a few number of galls,females as well as egg masses on roots of resistant tomatoes Agro-infected with TRV::map-1 and TRV::Cg-1 respectively.However,the numbers were much less than that produced on resistant tomatoes produced by the virulent population MIJS3-VI.These results showed that the silencing of map-1 and Cg-1 genes enhanced the reproduction ability of J2s of avirulent population MIJS3-AV on resistant tomatoes,which supported their relationship with avirulence/virulence of M.incognita.Conclusively,the results from in vitro and in vivo RNAi demonstrated that map-1 and Cg-1 genes were possibly linked with avirulence/virulence of M.incognita.However,silencing either map-1 or Cg-1 could not influence the avirulence/virulence of M.incognita completely,which revealed the variation in virulence of M.incognita was attributed by genetic quality instead of quantity.