Mechanism Of Meloidogyne Incognita Second-stage Juveniles Surviving Out Of Hosts

Plant pathogenic nematodes have exceeded bacteria and virus,ranking only second to pathogenic fungi on crop loss.Among common plant parasitic nematodes,Meloidogyne incognita widely distributes and induces most serious crop loss,having been the limiting factor of world agriculture sustainable development.Efficient and safe methods of control M.incognita are unavailable due to wide range of hosts and endoparasitism.M.incognita second-stage juveniles(J2),hatching from egg masses,need to overcome adversity prior to infestation of hosts and starvation is one of the most serious adversities J2 facing.Thus,accurate understanding of mechanism by which J2 live out of hosts on the basis of molecular level is the key and basics as for adoption of efficient and safe methods for controlling M.incognita.However,the mechanism by which J2 tolerate starvation remained unclear to date.Previous study had shown that lipid content decreased with aging increasing in the absence of hosts.However,it is obscured whether lipids breakdown is related to resistant to J2 starvation.On the basis of M.incognita genome information,this study slected three groups of J2 surviving out of hosts for different timing(namely Fresh,Middle and Last)for RNA-seq and found that differential expressed genes mainly enrichmented in these pathways including lipids metabolism and lysosome.The results of RNA-seq were further confirmed by lysosome specific dyeing and ultrastructural analysis.This study found that "arachidonic acid-lysosome-lipids" signal pathway cascades regulated J2 adipolysis in respond to starvation through RNA interference and drug inhibition,providing theory basis for research of controlling M.incognita.The main findings are as follows:1.Analysis of RNA-seq indicated that the number of differential expressed genes induced by short-term starvation was 1852,of which 952 was up-regulated and the number of differential expressed genes induced by long-term starvation was 1930,of which 862 was up-regulated.The number of shared differential expressed genes between short-term and long-term starvation 1124.Analysis of GO and KEGG enrichments of differential expressed genes implied that up-regulated genes induced by starvation mainly involved in lipids metabolism,amino acid metabolism,synthesis and decomposition of ketone bodies,lysosome pathway.Down-regulated genes indcued by starvation mainly engaged in glycometabolism and transport of substrates across membranes.Up-regulated genes enrichmented in lipids metabolism were homologs of lipase gene(atgl-1),fatty acid ? oxidative gene(acox-1),fatty acid desaturase gene(fat-6,fat-3)and hnf-4,ppar which both regulate lipid metabolism.As for those up-regulated genes involed in lysosomal pathway,most of them were genes encoding cathepsin,glycosidase and lipase.These suggested that J2 survived out of hosts,lysosomal biogenesis and lipids metabolism obviously enhanced.2.Starvation stress enhanced expression of Mi-hlh-30,encoding a transcription factor.J2 treated with Mi-hlh-30_dsRNA shown a decline in expression of Mi-lipl-1. Meanwhile,lysosomal biogenesis was depressed,survival time out hosts shortened,adipolysis was suppressed,motility declined.These implyied that Mi-hlh-30 regulate lysosomal biogenesis and adipolysis.3.This study found starvation stress elevated relative content of arachidonic acid Knock-down of Mi-fat-6 or Mi-fat-3 both involved in biosynthesis of arachidonic acid induced silence of Mi-hlh-30,yielding to depression of lysosomal biogenesis.Meanwhile,survival time,adipolysis,motility were suppressed.Supplement with downstream product,arachidonic acid,rescued phenotypic defects induced by the knock-down of Mi-fat-6 or Mi-fat-3 but not Mi-hlh-30,further confirming that Mi-fat-6 and Mi-fat-3 triggered expression of Mi-hlh-30 through regulating biosynthesis of arachidonic acid,resulting in lysosomal biogenesis.4.Treatment with lysosome specific inhibitors(chloroquine,ammonium chloride,Baf-Al)shown phenotypic defects resembling those induced by interference of Mi-fat-6,Mi-fat-3 or Mi-hlh-30,further confirmed the importance of lipophagy mediated by lysosome for J2 survival out of hosts.5.Found that Mi-RAB7 was involved in adipolysis of lipid droplets mediated by lysosome and knockdown of Mi-Rab7 depressed breakdown of lipids,leading to shorten of survival time out of hosts.In conclusion:1.Confirmed for the first time that lysosome was involved breakdown of lipids of J2 in vitro.2.Confirmed that starvation induced expressions of Mi-fat-6 and Mi-fat-3 to enhance expression of Mi-hlh-30 mediated by arachidonic acid,yeilding to biogenesis of lysosome and breakdown of lipids.