Study On The Analysis Of Functional Domain Of Peptidoglycan-associated Lipoprotein In Candidatus Liberibacter Asiaticus

Citrus Huanglongbing (HLB) caused by phloem-limited unculturable Gram-negative bacteria, is one of the most devastating disease worldwide. Peptidoglycan-associated lipoprotein (Pal) belongs to the bacterial outer membrane protein and plays a significant role in the pathogenicity of Gram-negative bacteria. Based on the previous research, this paper studies the depth of Pal gene of effector function in Candidatus Liberibacter asiaticus (CaLas), and clarified the expression characteristics of the gene in tobacco and Escherichia coli. The main results obtained in the present study were summarized as follows:1. Pal gene mutants phenotype analysis in Nicotiana benthamian: A sereies of point mutation and deletion mutants of Pal named ?Pal6th(aa7-161), ?Pal7th(aa8-161), ?Pal8th(aa9-161), ?Pal9th(aa10-161), ?Pal10th(aa11-161), ?Pal11th(aa12-161), ?Pal7th(Phe-Gly), ?Pal8th (Ile-Ala), ?Pal9th (Ile-Ala) and ?Pal10th (Leu-Gly) genes were constructed to entry vector pDONRTM/Zeo separately and then subcloned to plant expression vector pSK103 following the protocol of gateway technology. The recombinant plasmid containing corresponding gene was obtained and transformed into GV3101 and transiently expressed in N.benthamian. Among these constructs, only ?Pal6th(aa7-161)-103,?Pal7th(aa8-161)-103 and ?Pal7th(Phe-Gly) -103 were able to induce hypersensitive response in tobacco, Local necrosis was obvious at 6dpi, strong necrotic was observed at 12 dpi, But other deletion mutants and point-mutation mutants did not cause HR response , thus indicating the 8th amino acid Isoleucine (?e) in the transmembrane domain of Pal determined the HR reaction in tobacco.2. Subcellular localization analysis in N.icotiana benthamian: recombinant constructs of ?Pal7th(aa8-161)-Zeo, ?Pal8th(aa9-161)-Zeo, ?Pal7th (Phe-Gly)-Zeo and ?Pal8th (Ile-Ala)-Zeo in plant expression vector pMW390 were transiently expressed N. benthamian via Agrobacterium tumefaciend. At the time point of 48 hours post inoculation, samples were collected and observed under the fluorescent microscope. All the constructs of ?Pal7th(aa8-161)-390, ?Pal8th(aa9-161)-390, ?Pal7th (Phe-Gly)-390 and ?Pal8th (Ile-Ala)-390 gave strong fluorescent signal in cytoplasm membrane. And in rare cases, ?Pal7th(aa8-161)-390 and APal7th(Phe-Gly)-390 was observed fluorescent signal in nucleus, ?Pal8th(aa9-161)-Zeo and ?Pal8th(Ile-Ala)-Zeo was not observed fluorescent signal in nucleus.3. Callose deposition and oxidative burst in N.benthamian: ?Pal7th(aa8-161)-103, ?Pal8th(aa9-161)-103, ?Pal7th (phe-Gly)-103 and ? Pal8th (Ile-Ala)-103 were successfully constructed into pSK103. As observed under fluorescent microscope: ?Pal7th(aa8-161)-103 and ?Pal7th (Phe-GIy)-103 induced strong callose deposition in tobacco at the time point of 24-48 h post inoculation, But the accumulation of H2O2 was rapidly increased in 0.5-5 h after inoculation by DAB staining method, indicating that isoleucine play important role in the Pal function of triggering tobacco defense response.4. Functional analysis in pomelo: recombinant constructs of ?Pal6th(aa8-161)-103, ?Pal8th(aa9-161)-103, APal7th (Phe-Giy)-103 and ?Pal8th(Ile-Ala)-103 were transiently expressed in pomelo. ?Pal7h(aa8-161)-103 and ?Pal Pal7th (Phe-Giy)-103 gene constructs induced yellowing symptom near the inoculation point of pomelo, but other gene constructs and controls did not give similar symptoms. Further analysis by RT-qPCR showed that ?Pal7th(Phe-Giy)-103 and ?Pal Pal8th(Ile-Ala)-103 gene constructs could trigger different levels'defensive gene expression in pomelo. Such as the representive HR response genes like PR1, EDS1, PAL1, WRKY and NPR family genes were up-regulated by different level.?Pal7th(Phe-Gly)-103 gene constructs could trigger that the expression of PR1 was up-regulated 5 times at 8 dpi, 6 times of EDS 1 at 5 dpi, 1.5 times of WRKY 1 and PAL1 at 5 dpi and 4 times of NPR3 at 8 dpi. Compared with ?Pal7th(Phe-Gly)-103 gene constructs,?Pal8th(Ile-Ala) -103 gene constructs induced the HR marker gene PR1, NPR3 and EDS1 were different about 3 times at 5-8 dpi, so our results indicated that PTI immune reaction of pomelo was initiated.5. Subcellular localization analysis in E.coli: ?Pal7Th(aa8-161)-GFP, ?Pal8th(aa9-161)-GFP,?Pal Pal7th(Phe-Gly)-GFP and ?Pal Pal8th(Ile-Ala)-GFP was successfully constructed into the prokaryotic expression vector 1GFP using Linked Reactive Cloning (LIC) method. Under the condition of 28�C and 0.1 mM IPTG supplied, the recombinant plasmids could express target proteins successfully. All the above constructs were unipolar localized to the bacterial poles. Meanwhile the 1GFP empty vector construct was even distributed throughout the whole E. colif cell observed under laser scanning confocal microscope. The result indicated that the 8th isobaric amino acid of the ?PalCaLas gene in it's transmembrane domain affected the subcellular localizaiton in E.coli and maybe involved in the biological function in HLB bacteria.