Functional Analysis Of Holin_CLas Gene In Prophage Of "Candidatus Liberibacter Asiaticus"

Citrus Huanglongbing(HLB),is arguably the most damaging disease of citrus worldwide.HLB bacteria(CLas)is an unculturable phloem-limited α-proteobacteria.Due to its fastidious character,the pathogenicity of HLB bacteria is still not clear.In this study,the HolinCLas gene was selected from the genome of Candidatus Liberibacter asiaticus(CLas)and characterized by transient expression of pathogenic phenotype,subcellular localization and triggering of citrus defense response-related genes expression.The function of the gene was explored,and the main research results obtained were as follows:1.Analysis of the expression level of HolinCLas gene and the pathogenicity phynotype in samples showing different HLB symptoms:Citrus madurensis Lour from Fujian,Citrus grandis(L.).Osbeck.cv.guanxi miyou from Yunnan,and Citrus grandis var Shatian Yu Hort,Citrus maxima(Burm.)Merr.cv and Catharanthus roseus from Guangdong were collected and classified into different groups according to the symptoms.In the samples showing blotchy mottling,the expression level of HolinCLas gene was significantly higher than the others,followed by samples with yellow vein symptom.The expression level of HolinCLas was not detected by RT-PCR in the samples with severe blotchy mottling,even yellowing and nutrition deficiency symptoms.It indicated the expression level of HolinCLas gene in HLB different symptomatic samples varied greatly which laid the foundation for the next experiment.2.Pathogenicity phynotype analysis of transient expression of HolinCLas gene:The phenotype of HolinCLas gene transiently expressed in N.benthamiana.Necrosis was observed at the inoculation site at 4 dpi.Local necrosis was obvious at 6 dpi,and strong necrotic was observed at 12 dpi.It indicated that the HolinCLas gene induced hypersensitive response in N.benthamiana.The phenotype difference caused by the HolinCLas gene in different citrus varieties was observed after inoculation to Nobis Tangerine,C.reticulate Banco,C.sineses Osbeck cv,C.grandis var Shatian Yu Hort and C.limon(L.)Burmf via Agrobacterium.Compared to the control,yellowing of the infiltration area was observed at 5 dpi and slight necrosis appeared nearby the inoculation site after the transient expression in Nobis Tangerine.With the increasing of the inoculation time,the degree of yellowing and necrosis was aggravated,and the necrosis area expanded;a similar phenotype was observed on C reticulate Banco with slight yellowing compared with Nobis Tangerine.On C.sineses and C.limon,the obvious yellow with halo around the inoculation site was observed which became more serious in the late stage.Only the yellowing of the leaves was observed on C.grandis var Shatian Yu Hort,and don’t have yellowing halo near inoculation hole.3.Subcellular localization analysis of HolinCLas gene:HolinCLas was transiently expressed in N.benthamiana via Agrobacterium.Fluorescence signal was observed both in chloroplast and mitochandria,but no fluorescence signal was observed in non-inoculated tobacco or inoculated with empty vector pMW390.4.Citrus and tobacco defense response analysis:It induced a large accumulation of callose in 24-72 hpi.The accumulation of H2O2 and callose indicate that the HolinCLas gene can cause the defense response of N.benthamiana.The leaves of N.benthamiana and Nobis Tangerine can observed a large amount of starch accumulation in the infiltration range with iodine-starch staining.The expressions of defense-related genes of Nobis Tangerine,C.reticulate Banco,C.sineses Osbeck cv,C.grandis var Shatian Yu Hort and C.limon(L.)Burmf were analyzed by qRT-PCR.The gene PR 1,Plastocyanin,WRKY1,EDS 1 were up-regulated 3-13 times,and the WIPK gene was up-regulated 100-250 times,PR1 and Plastocyanin are up-regulated 13 times and 15times respectively.PR1,ICS1,WRKY1,EDS1,Bak1,and PAL1 were up-regulated 2.5-19 times in C.grandis var Shatian Yu Hort,the ICS1 gene which plays a key role in the salicylic acid synthesis is up-regulated 19 times at 8d.In C.sineses Osbeck cv,EDS1,NPR3,and PAL1 were up-regulated 1.5-4 times.ACRE31,EDS1,and PAL1 in C.limon(L.)Burmf were up-regulated by 1.5-2.8 times.The WIPK gene in C.sineses Osbeck cv,C.grandis var Shatian Yu Hort and C.limon(L.)Burmf was up-regulated at 2 days after inoculation.From the perspective of the change of defense gene expression,it was found that the HolinCLas gene can cause defense responses of different citrus varieties.5.Construction of prokaryotic expression vector and preparation of polyclonal antibody:In the induction of 1mmol/L IPTG at 37℃ for 4 h,a protein of about 20-25 kDa was obtained.The HolinCLas-28a plasmid was sent to company for polyclonal antibody preparation.CLas-specific,high titer polyclonal antibody was obtained.