Functional Analysis Of Tol-Pal Operon In Candidatus Liberibacter Asiaticus

Citrus Huanglongbing（HLB）is one of the most devastating disease in citrus production areas worldwide.The causal agent is an uncultivable phloem-restricted gram-negativeα-Proteobacteria.Tol-Pal operon plays crucial role in maintaining cell morphology and proliferation,as well as the pathogenesis in many gram-negative bacteria.Preliminary studies in our laboratory revealed that Pal _CLasacts as a PAMP,causing hypersensitive response,callose deposition and starch accumulation in tobacco.It may play an important role in triggering host plant resistance to the HLB bacteria.In order to further illustrate the function of the Tol-Pal operon on the morphology,proliferation and pathogenicity,Rm1021 was used as an alternative host of CLas.Knocking out mutants of theΔPal_Rm1021,ΔTol B_Rm1021,andΔTol Q_Rm1021genes,as well as the complementary strains ofΔPal_Rm1021/Pal _CLas::p BBR1MCS-5,ΔTol B_Rm1021/Tol B_CLas::p BBR1MCS-5 andΔTol B_Rm1021/Tol C_CLas::p BBR1MCS-5 were constructed.The main results are as follows:1.Construction of the knocking out and the complementary strains of the Pal,Tol B,and Tol Q in Rm1021:according to the principle of gene homologous recombination,we succeeded in construction of the knocking out mutants ofΔPal_Rm1021,ΔTol B_Rm1021,ΔTol Q_Rm1021,as well as the complementary strains ofΔPal_Rm1021/Pal _CLas::p BBR1MCS-5,ΔTol B_Rm1021/Tol B_CLas::p BBR1MCS-5 andΔTol Q_Rm1021/Tol Q_CLas::p BBR1MCS-5.2.Biological characteristics of knocking out mutants and the complementary strains:with the help of laser scanning confocal microscope and the transmission electron microscope,the difference in morphology ofΔPal_Rm1021and wild-type strains Rm1021 was observed:ΔPal_Rm1021 cells were significantly smaller than Rm1021;and the growth rate ofΔPal_Rm1021,ΔTol B_Rm1021,ΔTol Q_Rm1021 was slower than that of the wild type Rm1021,and there is no significantly difference among knocking out mutants.All the knocking out mutants entered the logarithmic growth phase after 36 h,and entered the growth quiescent phase after 52 h.The complementary strain ofΔTol B_Rm1021/Tol B _CLas::p BBR1MCS-5recovered the corresponding knocking out mutants best.And it entered the growth quiescent period 12h later than the wild type Rm1021.In terms of salt tolerance,with the increase of the concentration of Na Cl,the growth rate of the knocking out mutants,the complementary strains and the wild-type were slow down,and all the colonies became smaller.The complementary strains can basically restore the phenotype of the wild-type.In terms of motility,the swimming and swarming abilities of the knocking out strains were significantly reduced.For swimming ability,the swimming diameter of wild type Rm1021 was 0.87±0.21 cm,and the dissemination diameter ofΔTol B_Rm1021,ΔPal_Rm1021andΔTol Q_Rm1021was around 0.4±0.02-0.5±0.05 cm.There’s significant difference among each other（P=0.05）.All the complementary strains can restore the swimming ability to some extent compared to the wild-type.And no significant difference was observed among each other（P=0.05）.In terms of the swarming motility,except forΔTol B_Rm1021,no significant difference was observed as compared to the wild type（P=0.1）.All the complementary strains ofΔPal_Rm1021/Pal _CLas::p BBR1MCS-5,ΔTol B_Rm1021/Tol B_CLas::p BBR1MCS-5 andΔTol Q_Rm1021/Tol Q_CLas::p BBR1MCS-5 can restore the phenotype of the wild-type.As for the sensitivity to the antibiotics,when the concentration of penicillin was 5.0μg/m L,the wild type Rm1021 was more sensitive and the growth was completely inhibited;but the knocking out mutants can survive.When the concentration of ampicillin was 5.0μg/m L,both the wild type Rm1021 andΔTol B_Rm1021 mutant can survive,but the growth ofΔPal_Rm1021andΔTol Q_Rm1021 were completely inhibited.The results indicated that the knocking out strains were more sensitive to ampicillin than the wild type.And the growth status of the commentary strains on the plates with antibiotic were consistent with that of the wild type.3.The function of Pal _CLas overexpression strain on the influence of morphology and proliferation:the growth rate of the overexpression strain was lower than that of the wild type strain.The Pal _CLas overexpression strain entered the logarithmic growth phase after34h.And entered the growth quiescent phase at 48h,which was 6 hours later than the wild type.The strain morphology was also changed:the length was shortened,the width became wider,the aspect ratio,the circumference and the area were all reduced as compared to the wild type Rm1021.4.Localization of Pal _CLas gene in Rm1021:using fusion PCR,we constructed Pal _CLasexpression vector with EGFP green fluorescent protein gene.Triparental conjugation was used to transfer the construction into Rm1021.Observation under laser scanning confocal microscope revealed that the green fluorescence was distributed throughout the bacteria body without obvious specific area preference,which was different from the polarity distribution as shown in E.coli.5.Interaction of the key elements in Tol-Pal operon:Pal _CLas,Tol B_CLas,and Tol Q_CLasin the Tol-Pal operon of CLas were constructed into AD and BD vector for mutual verification.The results showed that:Pal _CLas and Tol B_CLastransformed colonies can grow on the defective medium of QDO（SD/-His-Leu-Trp-Ade）.It produced obvious blue colonies on the plate supplemented with X-α-Gal,which proveed that there was an interaction between the Pal _CLas and Tol B_CLas gene.No obvious reactions were observed among other genes.