Effect Of Candidate Effectors Of The Cereal Cyst Nematode(Heterodera Avenae)on Plant Defense

Cereal cyst nematode(CCN)Heterodera avenae is one of the most important cereal cyst nematodes(CCNs)in the world.This pathogen is distributed worldwide on cereal crops and causes severe damage.H.avenae is an obligate sedentary plant parasitic nematode that can deliver kinds of effectors into root tissue to facilitate plant parasitism.On the other hand,plants activate their own defenses to avoid parasitism.This study focused on effect of candidate effectors of H.avenae on plant defense to contribute to knowledge of H.avenae-plant interaction.51 candidate effectors of H.avenae were assessed for their ability to suppress programmed cell death triggered by the pro-apoptotic mouse protein BAX(BT-PCD)using transient expression assays in Nicotiana benthamiana,and we found that most of them could suppress BT-PCD.Among them,41 genes(80%of totally tested genes)suppressed BT-PCD;3 genes(6%of totally tested genes)could not suppress BT-PCD.In addition,7 genes(14%of totally tested genes)themselves could trigger plant defense,causing cell death or chlorosis of N.benthamiana leaf.Afterwards,we selected an annexin-like gene Ha-annexin for further function study.The gene encodes a predicted 326 amino acid protein and has highest similarity to annexin 2,which encodes a secreted protein of Globodera pallida.Southern blotting revealed that there are at least two homologies in the H.avenae genome.Ha-annexin transcripts were expressed within the subventral gland cells of the pre-parasitic second-stage juveniles by in situ hybridization.Additionally,expression of these transcripts were relatively higher in the parasitic second-stage juveniles by quantitative real-time RT-PCR analysis,suggesting the importance of the gene during early parasitism.Knockdown of Ha-annexin by method of barley stripe mosaic virus-based host-induced gene silencing(BSMV-HIGS)caused impaired nematode infections at 7 dpi and reduced females at 40 dpi,indicating important roles of the gene in parasitism at least in early stage in vivo.Transiently expression of Ha-ANNEXIN in onion epidermal cells and N.benthamiana leaf cells showed the localization at cytoplasm and plasma membrane.Further study revealed that Ha-ANNEXIN could suppress the induction of 3 marker genes(NbPti5,NbAcre31 and NbGras2)of PAMP-triggered immunity(PTI)in N.benthamiana,suggesting its suppression of PTI.In addition,Ha-ANNEXIN targeted a point in the mitogen-activated protein kinase(MAPK)signaling pathway downstream of two kinases MKK1 and NPK1 in N.benthamiana.In addition,as molecular biology and genetics experiments related to wheat are hard to conduct,an alternative model host for study of the plant-H.avenae interaction would be exceptionally useful.In this research,we assessed the sensitivity of 25 inbred lines of Brachypodium distachyon to H.avenae J2s from the Daxing District in Beijing City(DX).All lines of B.distachyon could be infected,but only 13 lines could reproduce cysts.The entire root system of the infected B.distachyon appeared smaller and the fibrous roots were shorter and less numerous.We showed that Koz-1 of B.distachyon reproduced more cysts and would be more suitable as a host.Also it could support the complete life cycles of 5 CCN geographical populations belonging to the Hal or Ha3 pathotype group.And a dose of 1000 J2s of CCN was sufficient for nematode infection.