Functional Analyses Of Effectors Chorismate Mutase And Venom Allergen-like Protein From Root-knot Nematode Meloidogyne Incognita

Root-knot nematodes(RKNs,Meloidogyne spp.)are plant sedentary endoparasites and cause over a billion dollars of yield losses worldwidely.During its life cycle,the mobile second stage juvenile(J2)of RKN penetrates root tips and establish permanent feeding site which is crucial for nematode to obtain nutrients and sustain growth and development.During the parasitism,RKNs secrete a large amount of effector proteins and introduce them into plant cells via stylet.These proteins help nematode penetrating and migrating in the root,suppressing the host defenses and modifying the normal host cells into the giant cells for feeding sites.In previous work carried out by our lab,the effector chorismate mutase(CM)genes Mi-cm-1 and Mi-cm-3,as well as the venom allergen-like protein(VAP)gene Mi-vap-2 was successfully cloned from M.incognita.In this study,the functions of Mi-cm-1,Mi-cm-3 and Mi-vap-2 in parasitism process of M.incognita were systematically investigated and the results are as following:1.Mi-CM-1 and Mi-CM-3 have chorismate mutase functionTo investigate whether M.incognita effector Mi-CM-3 has the chorismate mutase function in vivo,Mi-cm-1 and Mi-cm-3 were respectively complemented into the CM-deficient E.coli strain JP2261 which is a non-pathogen,as well as into the CM-deficient Xanthomonas oryzae pv.oryzae strain PX099A which is a plant pathogen.The CM-deficient strains of JP2261 expressing Mi-cm-1 and Mi-cm-3 succeeded in growing on the M9 medium with absence of phenylalanine.This result revealed that Mi-CM-3 also has enzyme function like Mi-CM-1 which had been reported previously.The knockout of Xoo-cm in strain PXO99A decreased its virulence to leaves of the host rice,while enhanced its ability in triggering hypersensitive response and transcription of resistant related genes on the leaves of non-host tobacco.Compared with the wild type strain PX099A,the complementation of CM-deficient PX099A with Mi-cm-1 and Mi-cm-3 respectively enhanced their virulence to the host rice,and reduced their ability in triggering hypersensitive response and transcription of resistant related genes in non-host tobacco.The results showed that Mi-CM-1 and Mi-CM-3 both have function of chorismate mutase and were related to virulence of pathogen.2.Mi-cm-1 and Mi-cm-3 play essential roles in parasitism of M.incognitaThe expression patterns of Mi-cm-1 and Mi-cm-3 in different life stages of M.incognita were analysed by RT-PCR and the results revealed that the high transcription of Mi-cm-1 occurred mainly at nematode early infection stage,while Mi-cm-3 was at the late parasitic stage during after feeding sites established.In vitro RNAi of the second stage juveniles(J2s)of M.incognita was carried out with uptake of dsRNA stimulated by resorcino.RT-qPCR detection revealed that the transcript levels of Mi-cm-3 and Mi-cm-1 showed a significant reduction in J2s after 24 h soaking in their respectively dsRNA solutions.The parasitism of J2s treated with Mi-cm-1 dsRNA soaking on tobacco plants was apparently decreased compared with that of J2s treated with gfp dsRNA soaking.However,the parasitism of J2s treated with Mi-cm-3 dsRNA soaking did not show differences from that of the control.A system of Tobacco rattle virus(TRV)mediated gene silencing was established for in vivo RNAi of Mi-cm-1 and Mi-cm-3.RT-qPCR detections showed that the transcript levels of Mi-cm-l and Mi-cm-3 both were reduced in nematodes after 14 days inoculation on TRV N.benthamiana lines.The number of galls and females produced on TRV::cm-3 lines showed a significant decrease compared with that on TRV::gfp line.However,TRV::cm-1 lines did not showed any significant difference.These results demonstrated that Mi-cm-1 plays a role in the early infection stage of M incognita,while Mi-cm-3 has a role in establishing or maintaining the feeding sites.3.Over-expressions of Mi-cm-1 and Mi-cm-3 in N.benthamiana affect the plant development and nematode parasitismTo investigate the effects of over-expressing Mi-cm-1 and Mi-cm-3 of M.incognita in N.benthamiana on plant development and nematode parasitism,the plant expression vectors pBI121-cm-1 and pBI121-cm-3 were constructed.The tobacco plants constitutively expressing Mi-cm-1 and Mi-cm-3 were generated with the help of Agrobacterium tumefaciens,respectively.The root systems of transgenic plants of Mi-cm-1 and Mi-cm-3 were apparently shorter than that of the wild type and gfp transgenic plants.The over-expression of Mi-cm-1 and Mi-cm-3 both inhibited the plant growth.After inoculation with M.incognita J2s,the numbers of root knots and females produced on Mi-cm-1 and Mi-cm-3 transgenic tobacco plants were significantly increased compared with that on control plants.The results showed that the over-expressions of Mi-cm-1 and Mi-cm-3 in tobacco affected the plant development and improved the nematode pathogenicity.4.Mi-CM-1 and Mi-CM-3 suppressed the accumulation of salicylic acid and thus affected the disease resistance of N.benthamiana triggered by pathogenThe effects of Mi-CM-1 and Mi-CM-3 on disease resistance of N.benthamiana were investigated by A.tumefaciens mediated transient expression.The transiently expression effects of Mi-cm-1 and Mi-cm-3 on inducing N.benthamiana PTI(PAMP-triggered immunity)marker genes triggered by flg22 was detected by RT-qPCR.The production of reactive oxygen species(ROS)was analyzed with Luminol method.The results showed that Mi-CM-1 and Mi-CM-3 did not alter the transcription of the PTI marker genes Pti5,Grans2 and Acre31 as well as the production of ROS triggered by flg22.The leaves of N.benthamiana transiently expressed Mi-cm-1 and Mi-cm-3 were respectively inoculated with X.oryzae pv.oryzae PXO99A,and the amount of salicylic acid(SA)accumulation in leaves was analyzed using HPLC.The results showed that Mi-CM-1 and Mi-CM-3 suppressed the SA accumulation triggered by the inoculation of PX099A.RT-qPCR detection also revealed that Mi-CM-1 and Mi-CM-3 suppressed the transcription of PR1 and PAL triggered by bacteria inoculation.These results revealed that Mi-CM-1 and Mi-CM-3 suppressed the plant resistance to pathogens through interfering SA accumulation.5.Exposure to dsRNA mediated by TRV leads to transcription up-regulation of Mi-vap-2 from M.incognita and promotion of pathogenicity in progenyMi-vap-2 of M.incognita,a gene encoding a venom allergen-like protein,was targeted by RNAi mediated by TRV.Compared to wild type line,a substantial up-regulation of Mi-vap-2 transcript was observed in juveniles at 7-days post-inoculation(dpi)collected from N.benthamiana agroinfiltrated with TRV::vap-2.This up-regulation of the targeted transcription did not impact on development of females or the production of galls and females on the TRV::vap-2 line.In a positive 16D10 control line,the transcript of Mi16D10 was knocked down in juveniles at 7-dpi from the TRV::16D10 line,resulting in a significant inhibition of nematode development.The up-regulation of Mi-vap-2 triggered by TRV-RNAi was inherited by the progeny of the nematodes exposed to dsRNA.Meanwhile,a substantial increase of Mi-VAP-2 expression in those progeny juveniles was revealed by the enzyme linked immunosorbent assay(ELISA).This caused an increase in the number of galls(71.2%)and females(84.6%)produced on seedlings of N.benthamiana when compared with the numbers produced by control nematodes.Up-regulation of Mi-vap-2 and its encoded protein therefore enhanced the pathogenicity of nematodes,suggesting that Mi-vap-2 may be required for the successful parasitism during the early parasitic stage of M.incognita.In summery,the effectors,chorismate mutase Mi-CM-1 and Mi-CM-3 as well as the venom allergen-like protein Mi-VAP-2,play key roles in parasitism of M.incognita.Mi-CM-1 has a role in early infection stage of nematode,while Mi-CM-3 has a role in establishing or maintaining feeding sites.Mi-CM-1 and Mi-CM-3 both suppressed the disease resistance of host through altering the SA biosynthesis in plant cells.Mi-VAP-2 also has an essential role in the early infection stage of M.incognita,and enhances nematode parasitism.