Functions Of Phloem Protein 2 (PP2)Gene CsPP2B15 In Response To Huanglongbing In Citrus

Citrus Huanglongbing(HLB),associated with‘Candidatus Liberibacter asiaticus'(CLas),is the most devastating disease in citrus.CLas is a gram-negative bacteria living exclusively in the sieve of phloem.HLB has caused serious yield losses in citrus-planting regions all over the world,and there is no effective control method.So it is necessary to exploit resistance genes to improve citrus HLB resistance.When CLas infection,excessive deposition of callose in sieve pores caused the phloem plugging to prevent pathogen spread.Phloem Protein 2(PP2)is one of the most abundant proteins in phloem.Studies have shown that CsPP2 genes may be involved in callose deposition and phlome plugging caused by CLas infection,but studies on their functions in citrus have rarely been reported.Here,expression characteristics of CsPP2 genes between the susceptible variety Jincheng orange(Citrus sinensis Osbeck)and the tolerant variety sour pomelo(C.grandis Osbeck)were investigated by qRT-PCR to screen important CsPP2 genes in response to CLas infection,and the functions of canditative genes were evaluated through citrus transformation method.The goal of this study provides theoretsical basis and new gene source for resistance molecular breeding.The principal research results are as follows:1.Screening and cloning of CsPP2 gene infected by CLasSix CsPP2 genes(CsPP2B15?CsPP2B2?CsPP2A?CsPP2B12-1?CsPP2B10?CsPP2B12-2)have been obtained through comparing expressions of 109 PP2 genes between Jincheng orange(Citrus sinensis Osbeck)and sour pomelo.Cloning and sequencing showed that six CsPP2 genes had typical PP2 domains.Expression characteristics of six CsPP2 genes were further investigated by qRT-PCR: The expression of CsPP2B2 was upregulated by CLas infecton in Jincheng orange,while no change was detected in the young leaf of sour pomelo.The expression of CsPP2 A was upregulated by CLas infecton in both the old leaf of Jincheng orange and sour pomelo,while no induced expression were found in the young leaf of sour pomelo.The expression of CsPP2B12-1 was significantly upregulated by CLas infecton in both the young midrib of Jincheng orange and sour pomelo.The expression of CsPP2B10 was upregulated by CLas infecton in the young leaf of Jincheng orange,while it was repressed in sour pomelo.The expression of CsPP2B12-2 was upregulated by CLas infecton in both the young midrib of Jincheng orange and sour pomelo.The expression of CsPP2B15 was significantly induced by CLas infecton in the young leaf of Jincheng orange,while its expression has no change in the young leaf of sour pomelo.Among the six genes,the expression level of CsPP2B15 induced by CLas was the highest.Young leaf and flush was believed as the early infection sites of CLas pathogen.Thus,our results suggest that CsPP2B15 play a role in the early stages of CLas infection and closely related to the susceptibility of citrus varieties.Therefore,CsPP2B15 was selected for further study.2.Cloning and expression of CsPP2B15 promoterA 992 bp promoter sequence were cloned,and the gene promoter elements were analyzed.Results showed that the promoter elements including multiple pathogen-induced cis-elements,such as W2 box,GT1 box,dof box,and G box.The gene promoter also contains cis-elements involved in plant adversity or hormone response,such as ABRE,AuxRR-core,and HSE.In order to determine the tissue-specific expression of CsPP2B15 promoter,the plant expression vector containing GUS reporter gene driven by CsPP2B15 promoter were constructed,and introduced into the Poncirus trifoliata(L.)Raf.by Agrobacterium-mediated transformation.Histochemical GUS and quantitative analysis showed that CsPP2B15 promoter had strong activity and strict vascular-specific expression pattern in citrus.This provides a good candidate for targeting expression of antibacterial constructs to battle citrus huanglongbing disease.3.Effect of overexpression of CsPP2B15 on CLas infection in transgenic citrusFurther to explore functions of CsPP2B15 in response to CLas,the plant expression vector containing CsPP2B15 gene driven by CaMV35 S promoter were constructed,and introduced into the Jincheng orange by Agrobacterium-mediated transformation.Twelve transgenic plants overexpressing CsPP2B15 were obtained through GUS histochemical staining and PCR analysis.The transgenic plants responding to CLas infection were evaluated by leaf disc grafting.Our results showed that the bacteria growth in transgenic plants was significantly increased,the accumulation of callose was significantly enhanced,and phloem was significantly thickened,when compared with wildtype.Our data indicated overexpressing CsPP2B15 accelerates pathogen spread and disease development in CLas-infected plant.Totally,we showed that CsPP2B15 is closely related to the susceptibility of citrus variety to HLB,and has positive role in favoring pathogen spread and promoting HLB disease symptoms in CLas-infected plant.Our study provides a potential candidate for the molecular breeding of citrus resistance to huanglongbing.