Study On Interaction Between Effector MBL1 From ’Candidatus Liberibacter Asiaticus’ With Citrus Papain-Like Cysteine Protease

Huanglongbing(HLB)is considered a "cancer" in citrus production.With its rapid global spread,it has caused huge economic losses to the citrus industry.The causal agents of HLB are phloem-limited ‘Candidatus Liberibacter spp.’,which can be transmitted by grafting and citrus psyllid.So far,it has not been cultured artificially,and the understanding of its pathogenic mechanism is very limited.Studying the functions of pathogenicity related genes and identification of their host targets is helpful to reveal pathogenic mechanisms of pathogenic bacteria.It has been reported that CLIBASIA＿00495 is a metal-dependent hydrolase encoded by‘Candidatus Liberibacter asiaticus’(CLas),which belongs to β-lactamase family,hence the name MBL1.MBL1 was up-regulated during the infection of Citrus by CLas,and its signal peptide was proved to be functional by alkaline phosphatase analysis,therefore MBL1 was speculated to play a virulence function during CLas infection.To explore how MBL1 functions during CLas infection,the host targets of MBL1 were screened,and a citrus papain-like cysteine protease(PLCP),was identified to interact with MBL1 in this study.Further phylogenetic analysis of citrus PLCP family was carried out to study the interaction between MBL1 and PLCP family members.The following results were obtained:1.The expression level of MBL1 during infection of citrus by CLas was much higher than that in psyllids verified by quantitative real-time PCR,and subcellular localization results showed that MBL1 was localized to the plant nucleus and cytoplasm.A citrus homolog of Arabidopsis aleurain-like protease(AALP),a member of PLCP family,was identified to interact with MBL1.And the interaction was validated by yeast two-hybrid,bimolecular fluorescence complementation,and co-immunoprecipitation.2.Thirty-three PLCP family members were identified from Citrus sinensis genome,which were divided into 9 subfamilies.And their locations on chromosomes,gene structures,conserved motifs,and promoter cis-acting elements were analyzed.In addition,expression pattern analysis revealed that four PLCP members from four subfamilies were up-regulated in Citrus sinensis after HLB infection.Subcellular localization showed that Cs SAG12-14,Cs CTB1 Cs RD21-2,Cs RD19-3 and Cs ALP1 were localized in both cytoplasm and nucleus,while Cs XBCP2 was only localized in the cytoplasm but not in the nucleus.Y2 H results indicated that MBL1 did not interact with other PLCP family members,but only interacted with AALP specifically.3.The AALP protein contains a signal peptide,an N-terminal autoinhibitory prodomain,and a C-terminal papain domain.To identify the critical interaction site between AALP and MBL1,Y2 H was used to validate the interaction between MBL1 and truncated mutants of AALP.The results showed that the papain domain of AALP interacted with MBL1 and that the presence of the autoinhibitory prodomain inhibited the interaction.4.To test whether MBL1 inhibit cysteine protease activity of citrus AALP,the prokaryotic expressed MBL1 protein was purified,and a preliminary DCG-04-based activity profiling of papain-like cysteine proteases was established,which provided a foundation for further testing the inhibition of plant PLCP activity by MBL1.In summary,MBL1,an effector from CLas up-regulated during infection of citrus,specifically targeted the citrus AALP and interacted with the cysteine protease domain of AALP.The PLCP family is involved in the plant immunity,and several members were highly expressed in response to HLB.To interact with MBL1,the AALP must cut the autoinhibitory prodomain and form the mature papain.This study revealed that MBL1 interfered with the normal physiological processes of host cells by interacting with citrus AALP,which provided a foundation for understanding the pathogenesis of HLB.