The Mechanism Of Interaction Between "Candidatus Liberibacter Asiaticus" Effector Protein SDE4405 And Autophagy Related Protein CsATG8c To Promote Pathogen Infection

Citrus Huanglongbing（HLB）is one of the most threatening citrus diseases and has caused huge losses to the global citrus industry.So far,it has occurred in more than 50 countries and regions in Asia,Africa and North and South America.In China,HLB was epidemic in 11 out of 20 citrus planting provinces.The pathogen of HLB is Gram-negative bacteria and is an obligate parasite of phloem.There are three species,"Candidatus Liberibacter asiaticus"（CLas）,"Ca.L.africanus"（CLaf）and "Ca.L.americanus"（CLam）,among which CLas is the most aggressive to citrus industry.Since pure culture of the pathogen of HLB is not available,The pathogenic mechanism of CLas is not perfect.The whole genome of CLas is about 1.23 Mb and do not contain the typical type Ⅲ or Ⅳ secretion system（T3SS or T4SS）.Instead,it had the type I secretion system（T1SS）and SEC-dependent secretion pathway,which enabled CLas to secrete effector proteins.Previous studies have shown that CLas effector protein SDE4405（CLIBASIA₀4405）can inhibit cell death triggered by the pro-apoptotic mouse protein BAX and the Phytophthora infestans elicitin INF1 in Nicotiana benthamiana.This study further dissected the influence of SDE4405 on pathogen infection,identified host target proteins interacting with SDE4405,and explored thermolecular mechanism of their interaction.1.SDE4405 promotes the proliferation of pathogenic bacteria by downregulating the expression of host defense-related genesSDE4405 transgenic citrus were successfully constructed via Agrob acteri a-m edi ated epicotyl transformation using hyperexpression vector pLGN-SDE4405.Phenotypic observation showed that SDE4405 transgenic citrus and wild-type（WT）citrus had no difference in plant height and leaf size.After grafted with CLas-infected branches,CLas was detected periodically for by PCR.The results showed CLas were detected earlier（20 days）in SDE4405 citrus than in WT citrus.Symptoms of yellowing leaves and leathery vein appeared at 30 days after grafting in SDE4405 transgenic citrus.These results indicate that effector protein SDE4405 is one of the key pathogenic factors of HLB.After inoculated with Xanthomonas citri subsp.citri 29-1（Xcc 29-1）,volcanic-like canker colonys were found earlier in SDE4405 transgenic citrus leaves.Colony growth experiments showed that the CFU of Xcc 29-1 in transgenic plants was significantly higher than that in WT citrus.N.benthamiana leaves expressing SDE4405 for 24 h were inoculated with Pseudomonas syringae pv.tomato DC3000 hopQ1-1（Pst DC3000 hopQ1-1）.The results showed that the necrosis symptoms of expressing SDE4405 leaves were significantly lighter than those of the control,and the CFU of Pst DC3000 was significantly more than that of the control group.These results indicated that expression of SDE4405 promoted pathogen proliferation in the host.RNA was extracted from citrus leaves before and 30 days after infection with CLas and used for detecting the expression of defense-related genes by RT-qPCR.Compared with WT citrus,the expression of defense-related genes in SDE4405 citrus was down-regulated in transgenic citrus before inoculation.With the establishment of CLas,the downregulation of genes is more significant.Transcriptome sequencing showed that the differentially expressed genes associated with biological stress were downregulated more significantly after infection than before infection.These results indicated that expression of SDE4405 inhibited host defense response.2.The effector protein SDE4405 interacts with the autophage related protein CsATG8cTo find host target proteins that interact with SDE4405,yeast two hybrid（Y2H）assays were carried out and 36 citrus candidate target proteins interacting with SDE4405 were screened.The interaction between SDE4405 and CsATG8c was verified by assays of Y2H,bimolecular fluorescent complementary（BiFC）,firefly luciferase complementation imaging（LCI）,co-immunoprecipitation（Co-IP）and in vitro GST fusion protein pull-down（GST-pull down）.Meanwhile,the experimental results of Y2H,BiFC and LCI showed that SDE4405 also interacted with the CsATG8 protein family and NbATG8 protein family.Through the bioinformatic analysis of SDE4405 nucleotide sequences by iLIR software,SDE4405 has an ATG8 interaction site（AIM）at 88-93 aa.AIM deletion mutant（SDE4405L）and alanine mutant（SDE4405W91A/L93A,SDE4405M）of SDE4405 were constructed and used for Y2H and BiFC experiments.The results showed that the interaction between SDE4405 and CsATG8c was interrupted after the absence of AIM SDE4405.After alanine mutation,the two proteins still interact with each other,indicating AIM is the key site of SDE4405-ATG8c interaction.Subcellular localization analysis showed that SDE4405 and CsATG8c were localized to the nucleus and cytoplasmic membrane,and colocalization analysis showed that their interaction did not change their localization.3.Interaction of SDE4405 and CsATG8c activate host autophagy and inhibit host defense responseElectron microscopy assays were performed using leaves（which were treated with vacuole protease inhibitors E-64d by 100 μM）of transgenic citrus and WT citrus,and the autophagosome structure in SDE4405 citrus was significantly more than WT citrus.The results of confocal microscopy observation showed that the CFP-NbATG8f-labeled autophagosome structure in co-expressing SDE4405+CsATG8c leaves was significantly more than in the control.Using GFP-CsATG8c as the marker,and it was found that more autophagosome structures were observed in the leaves co-expressing SDE4405 and CsATG8c compared with the leaves that co-expressed SDE4405L+CsATG8c and SDE4405 alone.The autophagic flow detection experiments showed that the number of autophagic structures in E-64d treated leaf cells was significantly higher than that in untreated cells,indicating that autophagic flow was fluent.The above results indicate that the interaction between SDE4405-CsATG8c promotes cell autophagy.ATG8 silent plants of N.benthamiana and citrus were successfully obtained via Tobacco rattle virus（TRV）-based VIGS.Leaves from silent and non-silent N.benthamiana were inoculated with Pst DC3000 after expressing SDE4405 and co-expressing SDE4405+CsATG8c for 24 h.The leaves of silent plants expressing SDE4405 had the most severe necrosis symptoms,while the leaves of non-silent plants expressing SDE4405+CsATG8c had almost no symptoms.There was no difference in leaf symptoms between SDE4405-expressing non-silent plants and silent plants co-expressing SDE4405+CsATG8c.Colony growth experiments showed the CFU of Pst DC3000 in non-silent plants was significantly higher than that of silent plants after co-expression of SDE4405+CsATG8c or SDE4405 alone.These results indicate that the interaction of SDE4405+CsATG8c promotes Pst DC3000 proliferation in N.benthamiana.Leaves of WT and silent citrus were inoculated with Xcc.After 9 days of inoculation,the volcanic colonies of Xcc in non-silent plants were significantly larger than those of silent plants.The leaves of proteins transient expression were inoculated with Xcc,and the results showed the CFU of Xcc in SDE4405+CsATG8c expressing leaves was significantly higher than that of SDE4405 alone and SDE4405 and GUS leaves.These results indicate that the interaction of SDE4405+CsATG8c promotes Xcc proliferation in citrus.In conclusion,the co-expression of SDE4405+CsATG8c promoted the proliferation of the pathogen in the host.Pst DC3000 was inoculated into leaves of silent and non-silent N.benthamiana,which have expressed SDE4405 for 24 h.Leaves were collected at 36 hpi and used for the expression detection of defense-related genes by RT-qPCR.The expression of defense-related genes in silent plants was significantly higher than that of non-silent plants,indicating that ATG8 was in response to the defense response inhibition mediated by SDE4405.In summary,CLas effector SDE4405 directly interacts with ATG8s and their interaction activates autophagy and suppresses plant defense to promote bacterial multiplication.These data illustrate the critical roles of CLas effector protein in manipulating autophagy and enrich the current understanding of the interaction between CLas and citrus.