A Potential Disease Susceptibility Gene CsLOB of Citrus Is Targeted by a Major Virulence Effector PthA of Xanthomonas citri subsp. citri

Dear Editor, Citrus bacterial canker (CBC), one of the most destructive diseases of citrus, has a severe economic impact on citrus industries in tropical and subtropical countries (Das, 2003Das A. Citrus canker: a review.J. Appl. Hortic. 2003; 5: 52-60Google Scholar). It has been widely known that Xanthomonas citri subsp. citri (Xcc) causes the CBC by transferring the type-III effectors (T3SEs) to the interior of the plant cell through type-III secretion system (T3S) to modulate transcription of host plants to trigger CBC disease (Schaad et al., 2006Schaad N.W. Postnikova E. Lacy G. Sechler A. Agarkova I.V. Stromberg P.E. Stromberg V.K. Vidaver A.M Emended classification of xanthomonad pathogens on citrus.Syst. Appl. Microbiol. 2006; 29: 690-695Crossref PubMed Scopus (154) Google Scholar). Pathotype A, the Asiatic form of Xcc, has a wide host range, is pathogenic on almost all citrus varieties, and contains a major virulence gene pthA encoding a T3S-secreted transcriptional activator-like effector (TALE) that promotes cellular hypertrophy (Al-Saadi et al., 2007Al-Saadi A. Reddy J.D. Duan Y.P. Brunings A.M. Yuan Q. Gabriel D.W All five host-range variants of Xanthomonas citri carry one pthA homolog with 17.5 repeats that determines pathogenicity on citrus, but none determine host-range variation.Mol. Plant Microbe Interact. 2007; 20: 934-943Crossref PubMed Scopus (68) Google Scholar). However, the susceptibility gene in citrus being responsible for the CBC and matching PthA of Xcc is largely unknown. Previous studies in other Xanthomonas-plant pathosystems have revealed that TALEs are translocated into plant nuclei, bind to TALE-specific DNA coding regions, and activate transcription of downstream genes for disease susceptibility or resistance (Chen et al., 2010Chen L.Q. Hou B.H. Lalonde S. Takanaga H. Hartung M.L. Qu X.Q. Guo W.J. Kim J.G. Underwood W. Chaudhuri B. et al.Sugar transporters for intercellular exchange and nutrition of pathogens.Nature. 2010; 468: 527-532Crossref PubMed Scopus (752) Google Scholar). TALEs contain a central domain of tandem, nearly identical 33- to 34-amino-acid repeats that confer DNA-binding specificity (Boch et al., 2009Boch J. Scholze H. Schornack S. Landgraf A. Hahn S. Kay S. Lahaye T. Nickstadt A. Bonas U. Breaking the code of DNA binding specificity of TAL-Type III effectors.Science. 2009; 326: 1509-1512Crossref PubMed Scopus (1714) Google Scholar). The number of repeats varies among TALEs, and the residues at the 12th and 13th positions are hypervariable and known as repeat-variable diresidues (RVDs). The RVDs bind to specific, single nucleotides and, consequently, determine specificity for disease susceptibility (S) or resistance (R) (Boch et al., 2009Boch J. Scholze H. Schornack S. Landgraf A. Hahn S. Kay S. Lahaye T. Nickstadt A. Bonas U. Breaking the code of DNA binding specificity of TAL-Type III effectors.Science. 2009; 326: 1509-1512Crossref PubMed Scopus (1714) Google Scholar). Within the host cell, TALEs bind DNAs at UPT (up-regulated by TALEs) boxes, but the UPT boxes bound by PthA (or PthA homologs) in citrus have not been revealed yet. Thus, identification of S gene in citrus triggered by PthA is of significant importance in citrus resistance breeding by editing UPT box of such the S gene bound by PthA. In this study, to find the potential S gene, we first applied a deletion mutagenesis to a hypovirulent strain (Xcc049) of Xcc (Ye et al., 2013Ye G. Hong N. Zou L. Zou H. Zakria M. Wang G. Chen G. tale-based genetic diversity of chinese isolates of the citrus canker pathogen Xanthomonas citri subsp. citri.Plant Dis. 2013; 87: 1187-1194Crossref Scopus (13) Google Scholar). Southern blot, using 2.3-kb SphI fragment of pthA as a probe, demonstrated that Xcc049 possesses four pthA homologs (Figure 1A). Here, we named them in the knockout order as talA, talB, talC, and talD (Figure 1A and Supplemental Table 1). Correspondingly, the wild-type Xcc049 and mutant strains containing 4, 3, 2, 1, and non pthA homologs were designated as Xcc049A (wild-type, containing talA,B,C,D), Xcc049B (∆talA), Xcc049C (∆talA,B), Xcc049D (∆talA,B,C), and Xcc049E (∆talA,B,C,D) (Figure 1A). Then, to investigate the role of pthA in pathogenicity independently, complemented strain (Xcc049E-PthA) of Xcc049E with pthA (U28802.1) originating from X. citri subsp. citri strain 3213 (Swarup et al., 1992Swarup S. Yang Y. Kingsley M.T. Gabriel D.W A Xanthomonas citri pathogenicity gene, pthA, pleiotropically encodes gratuitous avirulence on nonhosts.Mol. Plant Microbe Interact. 1992; 5: 204-213Crossref PubMed Scopus (155) Google Scholar) was made in order that there is no other pthA homolog interference. The pathogenicity assay showed PthA not only restored, but also increased, pathogenicity and levels of growth in planta of Xcc049A in Citrus sinensis that exceeded the wild-type whereas Xcc049D and Xcc049E exhibited reduced pathogenicity and bacterial growth in planta, suggesting that talC is critical for CBC development in Xcc049A (Figure 1, Figure 1). RNA-seq is becoming a powerful tool to investigate the transcriptional profiles of some species samples stimulated by biotic or abiotic stresses (Wang et al., 2009Wang Z. Gerstein M. Snyder M. RNA-Seq: a revolutionary tool for transcriptomics.Nat. Rev. Genet. 2009; 10: 57-63Crossref PubMed Scopus (7380) Google Scholar). We sought to use RNA-seq to reveal potential target genes of C. sinensis activated by PthA, TalA, TalB, TalC, and TalD. For this, RNAs were isolated from citrus leaf regions, where six strains (Xcc049A, Xcc049B, Xcc049C, Xcc049D, Xcc049E, Xcc049E-pthA, and Xcc049A-pthA, containing four pthA homologs and pthA) were inoculated, while water-treated citrus was used as a control. The RNA-seq transcriptomes of the above-mentioned samples were generated by Majorbio Company (www.majorbio.com, Shanghai, China). To find citrus genes targeted by Xcc TALEs, three approaches were performed to identify the potential TALE targets. In the first approach, we compared Xcc049A to CK (water), Xcc049B to CK, Xcc049C to CK, Xcc049D to CK, Xcc049E to CK, and Xcc049E-PthA to CK. In the second, we compared Xcc049B to Xcc049A, Xcc049C to Xcc049B, Xcc049D to Xcc049C, Xcc049E to Xcc049D, and Xcc049E-PthA to Xcc049E. These two approaches were boosted to analyze the differential expression profiles of citrus genes activated by TALEs. The third one, Xcc049A-PthA to Xcc049E, was to predict the target sequences (UPT box) by PthA and the other PthA homologs of Xcc049A strain based on the code as described in the previous study (Boch et al., 2009Boch J. Scholze H. Schornack S. Landgraf A. Hahn S. Kay S. Lahaye T. Nickstadt A. Bonas U. Breaking the code of DNA binding specificity of TAL-Type III effectors.Science. 2009; 326: 1509-1512Crossref PubMed Scopus (1714) Google Scholar). The parameters for these three comparisons are log(Fold Change) ≥ 2 and P ≤ 0.05. From the consequence of the third approach we used, in total, 108 up-regulated genes were found (Supplemental Dataset 1). Then, using the genome database of ‘The Citrus sinensis Annotation Project’ (http://citrus.hzau.edu.cn/orange/index.php), we obtained all the promoters of these 108 genes (Supplemental Dataset 2). To identify the potential UPT boxes, two online programs (Target Finder https://tale-nt.cac.cornell.edu/node/add/talef-off and Talgetter http://galaxy2.informatik.uni-halle.de:8976/root?tool_id=TALgetter) (Doyle et al., 2012Doyle E.L. Booher N.J. Standage D.S. Voytas D.F. Brendel V.P. VanDyk J.K. Bogdanove A.J TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction.Nucleic Acids Res. 2012; 40: W117-W122Crossref PubMed Scopus (422) Google Scholar; Grau et al., 2013Grau J. Wolf A. Reschke M. Bonas U. Posch S. Boch J. Computational predictions provide insights into the biology of TAL effector target sites.Plos Comput. Biol. 2013; 9: e1002962.Crossref PubMed Scopus (72) Google Scholar) were used to search the promoter sequences by inputting RVDs of TalA, TalB, TalC, TalD, and PthA (Figure 1D and Supplemental Table 1). Intriguingly, both programs generated identical results, and revealed that the promoter of gene Cs7g27640 which got a score much higher than other predicted targets contained a UPT box that was predicted to match both PthA and TalC (KF682432) (Figure 1D). To confirm that the gene Cs7g27640, encoding a lateral organ boundaries (CsLOB) domain containing protein, is the target activated by PthA and TalC, we checked the transcription pattern of this gene in our first two comparison approaches mentioned above. The results showed that the gene was highly expressed when citrus leaves were inoculated with Xcc strains containing either PthA or TalC, and the expression level decreased dramatically in the absence of PthA or TalC (Figure 1E and Supplemental Datasets 3 and 4). To support this novel finding, we then artificially synthesized a dTALE that binds the upstream of the PthA UPT box of CsLOB (Supplemental Text 1, Supplemental Methods, and Supplemental Text 2) to investigate whether the dTALE acts as PthA or TalC to activate the expression of CsLOB for CBC symptoms. Indeed, the strain Xcc049E-dTALE triggered CBC disease as severe as that by Xcc049E-PthA (Figure 1F), strongly supporting that CsLOB is a potential target gene activated by PthA or TalC. We validated this target by fusing the 500-bp promoter of CsLOB to the promoterless gus gene. GUS activity was determined in the presence or absence of PthA or TalC in planta using Agrobacterium transient expression (Supplemental Methods). A promoter of CsSweet1(Cs3g20720), encoding a sugar transporter protein targeted by some known TALEs (Chen et al., 2010Chen L.Q. Hou B.H. Lalonde S. Takanaga H. Hartung M.L. Qu X.Q. Guo W.J. Kim J.G. Underwood W. Chaudhuri B. et al.Sugar transporters for intercellular exchange and nutrition of pathogens.Nature. 2010; 468: 527-532Crossref PubMed Scopus (752) Google Scholar), which was highly induced according to RNA-seq data (Supplemental Dataset 1) but was not predicted as the target gene by the two online programs (Supplemental Table 2), was used here as a control sequence (Figure 1D). Both PthA and TalC indeed triggered the transcriptional activation of GUS under CsLOB promoter, but not CsSweet1 (Figure 1G). The lower GUS activity induced by TalC than PthA shows that TalC has a weaker binding or/and activation ability (consistent with RNA-seq results; Figure 1E) which might be due to the different lengths of their RVDs (Morbitzer et al., 2010Morbitzer R. Roemer P. Boch J. Lahaye T. Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors.Proc. Natl Acad. Sci. U S A. 2010; 107: 21617-21622Crossref PubMed Scopus (200) Google Scholar). We hypothesize that the predicted inability of TalC to bind TATA box in the target UPT box sequence of CsLOB could be another reason (Figure 1D). This lower activity may explain why Xcc049A could not cause as much CBC disease or grow as well in planta as Xcc049A derivatives carrying PthA (Figure 1, Figure 1). These results indicate that PthA and TalC are transcriptional activators of CsLOB (Figure 1G). Our results also suggest that other highly induced genes identified by RNA-seq but without predicted UPT boxes would not be the direct targets. These genes may possibly be involved in CBC development particularly when CsLOB is activated by either PthA or TalC. This is worthy of further investigation. The identification of potential citrus susceptibility gene matching PthA could be useful for a variety of purposes. For example, targeted deletion of the UPT box by using the TALEN or CRISPR-Cas system would probably make the eradication of CBC possible (Feng et al., 2013Feng Z. Zhang B. Ding W. Liu X. Yang D.-L. Wei P. Cao F. Zhu S. Zhang F. Mao Y. Efficient genome editing in plants using a CRISPR/Cas system.Cell Res. 2013; 23: 1229-1232Crossref PubMed Scopus (555) Google Scholar; Joung and Sander, 2013Joung J.K. Sander J.D INNOVATION TALENs: a widely applicable technology for targeted genome editing.Nat. Rer. Mol. Cell. Bio. 2013; 14: 49-55Crossref PubMed Scopus (909) Google Scholar). We anticipate in the near future that the genome-modified resistance citrus lines will be generated and bring gigantic benefits to our citrus industry worldwide. Supplementary Data are available at Molecular Plant Online. This work was supported by grants from the National Science Foundation of China (31230059), the Ministry of Science and Technology (973 Program 2012CB114003), and the Special Fund for Agro-Scientific Research in the Public Interest (201003067-09).