The TALE effector PthA4 of Xanthomonas citri subsp.citri indirectly activates an expansin gene CsEXP2 and an endoglucanase CsEG1 via CsLOB1 to cause citrus canker symptoms

The TALE effector PthA4 of Xanthomonas citri subsp. citri indirectly activates an expansin gene CsEXP2 and an endoglucanase CsEG1 via CsLOB1 to cause citrus canker symptoms | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The TALE effector PthA4 of Xanthomonas citri subsp. citri indirectly activates an expansin gene CsEXP2 and an endoglucanase CsEG1 via CsLOB1 to cause citrus canker symptoms Rikky Rai , View ORCID Profile Nian Wang doi: https://doi.org/10.1101/2025.03.20.644280 Rikky Rai 1 Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida , Lake Alfred, FL, USA 2 Department of Botany, University of Allahabad , Prayagraj, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nian Wang 1 Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida , Lake Alfred, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nian Wang For correspondence: nianwang{at}ufl.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is an important citrus disease worldwide. PthA4 is the most important pathogenicity gene of Xcc and encodes a transcription activator like effector (TALE) secreted by the type III secretion system. PthA4 is known to activate the expression of CsLOB1 , the canker susceptibility gene and a transcription factor, to cause citrus canker symptoms. Extensive effort was made to identify downstream targets of CsLOB1 to investigate the mechanism underlying canker symptom development. However, none of identified CsLOB1 target genes have been confirmed to be involved in citrus canker development. Here, we first identified the direct targets of CsLOB1 by generating promoter- uidA (GUS) reporter fusion construct for the 13 genes highly induced by both PthA4 and CsLOB1 and monitored the reporter activity in N. benthamiana leaves co-expressing CsLOB1 . Agrobacterium tumefaciens -mediated transient expression of CsLOB1 activated seven gene promoters in N. benthamiana including Cs7g18460, Orange1.1t00600, Cs6g17190, Cs7g32410 ( CsEXP2 ), Cs2g27100, Cs2g20750 ( CsEG1 ), and Cs9g17380. Next, we constructed dTALEs to target unique sequences in the promoters of the seven direct target genes of CsLOB1 and transformed them into Xcc pthA4 ::Tn5 mutant. Our results indicate that a combination of 5 and 7 dTALEs caused canker-like symptoms in the inoculated citrus leaves. In addition, dTALECsEXP2 and dTALECsEG1 caused water soaking and pustules, which are typical canker symptoms. Taken together, Xcc indirectly activates CsEXP2 and CsEG1 via PthA4-CsLOB1 to cause canker symptoms. Author summary Ptha4 is responsible for the canker symptoms of Xcc and activate the expression of CsLOB1 of citrus to cause the canker symptoms. Extensive effort by multiple groups has been made to identify downstream targets of CsLOB1 to understand the mechanism underlying canker symptom development. However, none of the CsLOB1 targets have been shown to cause canker symptoms. Here we have demonstrated that Xcc indirectly activates CsEXP2, an expansin gene, and CsEG1, an endoglucanase, via PthA4-CsLOB1 to cause canker symptoms. Identification of direct targets of CsLOB1 provides alternative target genes for genetic improvement of citrus against canker via genome editing. Introduction Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most severe citrus diseases worldwide. It is endemic in most citrus production regions including Brazil, China, India, Mexico, USA except Australia and the Mediterranean region [ 1 ]. PthA4, the most important pathogenicity factor of Xcc, is a transcription activator like effector (TALE) which is secreted by the type III secretion system. PthA4 is responsible for the canker symptom development including water soaking, hypertrophy and hyperplasia and further release and spread from the lesions [ 2 , 3 ]. PthA4 binds to the promoter region (effector binding element (EBE)) to activate the expression of CsLOB1 , the canker susceptibility gene, thus causing canker symptoms [ 4 ]. Natural variations in the EBE region of LOB1 gene in Atalantia buxifolia contributes to its resistance against Xcc [ 5 ]. Similarly, TALEs have been known to activate disease susceptibility genes including OsSULTR3 :6 [ 6 ], UPA20 [ 7 ], and SWEET [ 8 , 9 ] to promote pathogen growth or symptom development in many pathosystems. Diverse approaches have been developed to control plant diseases by targeting the susceptibility genes. Genome editing of the EBE region of susceptibility genes has been widely used for engineering disease resistant crops. For instance, genome editing of the EBE of SWEET genes has led to the development of disease resistant rice varieties against bacterial blight [ 10 , 11 ]. Genome editing of the EBE region or TATA box of CsLOB1 genes has increased disease resistance against citrus canker [ 12 – 19 ]. Gene silencing of CsLOB1 via both transgenic approach [ 20 ] and application of antisense oligonucleotide [ 21 ] increase disease resistance against citrus canker. Importantly, non-transgenic genome editing of the EBE of CsLOB1 has led to the development of canker resistant citrus varieties that have been approved for commercialization by two federal regulatory agencies The Animal and Plant Health Inspection Service (APHIS) and Environmental Protection Agency (EPA) in the USA [ 18 , 22 – 24 ]. CsLOB1 belongs to the lateral organ boundaries domain (LBD) family transcription factors [ 25 , 26 ]. LBD proteins have diverse functions in plant growth and development [ 27 ] including in lateral root formation [ 28 ], and microspore division [ 29 ]. Extensive effort has been made to identify downstream targets of CsLOB1 to understand the mechanism underlying canker symptom development. Duan et al. conducted bioinformatics and electrophoretic mobility shift assays and showed that CsLOB1 binds to the promoter of Cs2g20600, a zinc finger C3HC4-type RING finger gene [ 30 ]. Zou et al. identified 565 putative CsLOB1-targeted genes via chromatin immunoprecipitation-sequencing (Chip-seq) [ 20 ]. de Souza-Neto et al. reported that CsLOB1, but not PthA4, directly actives the expression of an expansin gene CsLIEXP1 (orange1.1t00187) [ 31 ]. Long et al. showed that CsLOB1 promoted the expression of CsNCED1 -1, which encodes 9-cis-epoxycarotenoid dioxygenase, an enzyme in abscisic acid biosynthesis, by binding to its promoter. Chen et al. reported that CsLOB1 binds to the promoter of Cs9g12620, which encode a carbohydrate-binding protein [ 32 ]. In addition, it was reported that CsLOB1 activates a cellulase gene CsCEL20 by targeting its promoter region [ 33 ]. However, none of the previous studies have established that the identified CsLOB1 target genes are involved in citrus canker development. In this study, we cracked this puzzle using designer TALEs to activate putative CsLOB1 target genes. We have shown that CsLOB1 directly activates seven genes and activation of Cs7g32410 (hereafter CsEXP2 ), which encodes an expansin protein, and Cs2g20750 (hereafter CsEG1 ), which encodes an endoglucanase protein, using designer TALEs, led to canker symptoms. Results Identification of candidate direct target genes of CsLOB1 To identify the candidate target genes of CsLOB1 that are involved in causing citrus canker, we reasoned that genes whose expression was dependent on the presence of PthA4 from Xcc or CsLOB1 of citrus are likely to be regulated by CsLOB1. To identify such genes, we analyzed gene expression datasets and selected 14 candidate genes that are commonly and highly upregulated by both PthA4 and CsLOB1 [ 4 , 20 , 25 , 34 ] including Cs1g16560, Cs7g32410, Cs6g17190, Cs2g27100, Cs9g17380, Cs2g27090, Cs3g18550, Cs2g21440, Cs7g18460, Cs5g33680, orange1.1t00600, Cs2g05560, Cs2g20750, and Cs2g20600 ( Supplementary Table 1 ) [ 4 , 20 , 25 , 34 ]. View this table: View inline View popup Download powerpoint Supplementary Table 1. Genes positively regulated by PthA4 and CsLOB1 Seven candidate genes displayed CsLOB1-dependent promoter activity To examine if these 14 genes were directly regulated by CsLOB1, we amplified their promoter regions (approximately 1 kb in length) and checked their promoter activity in the presence or absence of CsLOB1 as described previously [ 31 ]. We were unable to amplify the promoter region of Cs3g18550 despite multiple attempts, so we proceeded with the promoter regions of the 13 other genes. We generated promoter- uidA (ß-glucuronidase (GUS) reporter fusion construct for the 13 promoters and monitored the reporter activity in N. benthamiana leaves transiently expressing CsLOB1 or EV (control) ( Fig.1 ) [ 31 ]. The 13 promoter::GUS fusions (pCs1g16560:: uidA , pCs7g32410:: uidA , pCs6g17190:: uidA , pCs2g27100:: uidA , pCs9g17380:: uidA , pCs2g27090:: uidA , pCs2g21440:: uidA , pCs7g18460:: uidA , pCs5g33680:: uidA , orange1.1t00600:: uidA , pCs2g05560:: uidA , pCs2g20750:: uidA , and pCs2g20600:: uidA ) were individually co-expressed in N. benthamiana with the 35S::CsLOB1-HA plasmid by Agrobacterium -mediated transformation ( Fig.1 B). As a positive control, the empty vector with 35S promoter upstream of uidA in pCAMBIA1380 was co-expressed in N. benthamiana in the presence or absence of CsLOB1 ( Fig.1C ). The co-inoculation of the 35S promoter fused to GUS resulted in no significant change (P ≤ 0.05) in GUS expression in the presence or absence of the CsLOB1 ( Fig. 1D ). Our results showed that A. tumefaciens -mediated transient ectopic expression of CsLOB1 activated seven gene promoters in N. benthamiana at 48 hours post inoculation (hpi) ( Fig.1D ) including Cs7g18460, Orange1.1t00600, Cs6g17190, Cs7g32410, Cs2g27100, Cs2g20750, and Cs9g17380, but not the six other genes. Cs7g18460, Orange1.1t00600, Cs6g17190, Cs7g32410, Cs2g27100, Cs2g20750, and Cs9g17380 encode glucomannan 4-beta-mannosyltransferase 2, 3-oxo-5-alpha-steroid 4-dehydrogenase, gibberellin-regulated protein 5, expansin, GDSL esterase/lipase, endoglucanase 11, and uncharacterized protein, respectively ( Supplementary Table 1 ). These results indicate that CsLOB1 directly induces the expression of these seven genes in N. benthamiana by potentially targeting an EBE site in their promoter regions. The seven promoters were searched for the 6-bp LBD motif (GCGGCG), which is known to bind to LOB domain proteins [ 35 , 36 ]. Among the 7 induced promoters by CsLOB1, Cs6g17190, Cs9g17380, Orange1.1t00600 but not Cs7g18460, Cs7g32410, Cs2g27100 and Cs2g20750 contain the LBD motif, suggesting other motifs can interact with LOB domain proteins. Download figure Open in new tab Fig. 1. Identification of genes directly regulated by CsLOB1. A. Schematic diagram of reporter and CsLOB1 expression constructs. Reporter constructs contained the promoter of genes of interest fused to the GUS reporter. CsLOB1 expression construct is driven by the 35S promoter and fused to HA tag. B. Schematic representation of Agrobacterium mediated inoculation of reporter and expression construct in Nicotiana benthamiana ( Nb ) leaves to measure the promoter activity in the presence or absence of CsLOB1. C. Detection of CsLOB1 expression in Nb leaves by western blotting. D. GUS assay. Samples were collected 48 hpi and GUS activity was measured three times with similar results. Promoters of the seven genes (Cs7g18460, Orange1.1t00600, Cs6g17190, Cs7g32410, Cs2g27100,Cs2g20750, and Cs9g17380) showed induction when CsLOB1 was ectopically expressed compared to empty vector (EV). The 35S promoter fused with GUS was used as a control. Error bars indicate means ± SD ( n = 3), and asterisks indicate significant differences (∗ P ≤ 0.05; ∗∗ P ≤ 0.01). Artificial dTALEs activate the expression of the identified CsLOB1 targets and trigger canker symptoms Next, we tested whether activation of Cs7g18460, Orange1.1t00600, Cs6g17190, Cs7g32410, Cs2g27100, Cs2g20750, or Cs9g17380 can cause citrus canker symptoms. For this purpose, designer TALEs (dTALEs) have been used to activate genes of interest specifically [ 37 , 38 ]. Accordingly, we constructed dTALEs to target unique sequences in the promoters of the seven direct target genes of CsLOB1 to induce each gene individually using optimized repeat variable di-amino acid (RVD) residues ( Fig. 2 ). The dTALEs were cloned into a pBBR5 vector with a C-terminal HA-tag epitope and introduced into Xcc306 pthA4 ::Tn5 and tested whether they can cause citrus canker symptoms on citrus leaves. Western blot analysis confirmed that the dTALEs were expressed in Xcc ( Fig.2C ). RT-qPCR assays showed that Cs2g27100, Cs7g18460, Orange1.1t00600, Cs6g17190, Cs7g32410, and Cs2g20750 genes were significantly induced by their respective dTALEs whereas Cs9g17380 induction was not statistically significant ( Fig.3 ). In all cases, the 7 genes were highly induced in the positive controls Xcc WT and pthA4 ::Tn5 mutant transformed with dTALECsLOB1 ( Fig. 4 ). We also checked whether dTALEs targeting genes of interest impact the expression of CsLOB1 by RT-qPCR. Expectedly, CsLOB1 was activated by Xcc WT and dTALECsLOB1 compared to the pthA4 mutant. None of the dTALEs targeting the promoters of the 7 genes induced the expression of CsLOB1 , eliminating the potential interference from CsLOB1 ( Fig. 4 ). Download figure Open in new tab Fig. 2. Activation of putative CsLOB1 target genes using designer TALEs (dTALEs). A. RVD repeat arrays of dTALEs used to activate the target genes. B. dTALE constructs were digested from pTAL2 destination vector, subcloned into pBBR5 with an HA tag at C-terminal. C. Detection of expression of dTALEs using Western Blotting. Total protein was extracted from overnight cultures of Xcc pthA4 :Tn5 [Negative control (NC)], and Xcc pthA4 :Tn5 transformed with the dTALE targeting CsLOB1 as positive control and dTALEs targeting other genes. Samples were separated by SDS PAGE and immunoblotted with the anti-HA antibody. Multiple lanes under the black line indicate independent colonies transformed with the respective dTALEs constructs. Download figure Open in new tab Fig. 3. Confirmation of the expression of putative CsLOB1 target genes driven by dTALEs. A, A schematic representation of Valencia sweet orange leaf infiltrated with Xcc strains WT, Xcc pthA4 ::Tn5 mutant, and Xcc pthA4 ::Tn5 strains carrying dTALE CsLOB1 or dTALE gene of interest using a needleless syringe. B. Expression analysis of CsLOB1 , Cs2g27100 , Cs7g18460 , Orange1.1t00600 , Cs9g17380 , Cs6g17190 , Cs7g32410 , and Cs2g20750 in sweet orange leaves infiltrated with a combination of Xcc pthA4:: Tn5 strains carrying the pBBR5 constructs dTALECs2g27100, dTALECs7g18460, dTALEOrange1.1t00600, dTALECs9g17380, dTALCs6g17190, dTALECs7g32410, and dTALECs2g20750. Xcc pthA4: :Tn5 and Xcc pthA4 ::Tn5 dTALECsLOB1 served as negative and positive controls, respectively. Sweet orange leaves were infiltrated and collected at 7 dpi for gene expression assay using RT-qPCR. Error bars indicate means ± SEM ( n = 3), and asterisks indicate significant differences in the expression of genes (∗ P ≤ 0.05) compared to that of Xcc pthA4: :Tn5 mutant. The results shown are representative of three independent replicates. Download figure Open in new tab Fig. 4. Expression analysis of CsLOB1 in Sweet orange leaves infiltrated individually with Xcc or Xcc PthA4:: Tn5 carrying the pBBR5 constructs for dTALECsLOB1, dTALECs2g27100, dTALECs7g18460, dTALECs2g20750, dTALECs7g32410, and dTALECs9g17380. pthA4 ::Tn5 served as negative control. Sweet orange leaves were infiltrated and collected at 2 dpi for gene expression assays using RT-qPCR. Error bars indicate means ± SEM ( n = 3), and asterisks indicate significant differences in the expression of CsLOB1 (∗ P ≤ 0.05) compared to pthA4: :Tn5 mutant. The results shown are representative of three independent replicates. To test whether dTALEs targeting the 7 CsLOB1 direct target genes can cause canker like symptoms, fully expanded young leaves of sweet orange were infiltrated with Xcc WT, pthA4 ::Tn5 mutant, dTALECsLOB1 and dTALEs at the concentration of OD600 of 0.2. First, we co-expressed a combination of 5 dTALEs (Cs2g20750, Cs6g17190, Cs7g32410, Cs7g18460, Orange1.1t00600) and 7 dTALEs (5 dTALEs plus dTALECs2g27100 and dTALECs9g17380) to comprehend if these dTALEs can cause canker like symptoms. Our results indicate that a combination of 5 and 7 dTALEs caused canker-like symptoms ( Fig. 5 ). Next, we tested whether individual dTALEs can cause canker symptoms. Interestingly, dTALECs7g32410 and dTALECs2g20750 caused water soaking and pustules, dTALECs7g18460 and dTALECs6g17190 caused slightly yellowing, and the other three dTALEs did not cause any symptoms ( Fig. 6 ). Download figure Open in new tab Fig. 5. Virulence assay with constructed dTALEs in Sweet orange leaves: Xcc strains WT, Xcc pthA4 ::Tn5, Xcc pthA4 ::Tn5 dTALECsLOB1 and Xcc pthA4 ::Tn5 dTALEs for putative CsLOB1 target genes were infiltrated into fully expanded young leaves of Valencia sweet orange, which were evaluated for the symptoms at 14 and 21 dpi. Representative results were chosen from three independent experiments. A. Schematic representation of inoculation of leaves with different strains. B. Combination of 5 dTALEs shown with black dotted circle (Cs2g20750, Cs6g17190, Cs7g32410, Cs7g18460, Orange1.1t00600). C. Combination of 7 dTALEs shown with black dotted circle (Cs2g20750, Cs2g27100, Cs7g32410, Cs7g18460, Orange1.1t00600, Cs9g17380, Cs6g17190. D. mock control. Download figure Open in new tab Fig. 6. Virulence assay with dTALEs in Sweet orange leaves. Xcc strains WT, Xcc pthA4 :: Tn5, Xcc pthA4 ::Tn5 dTALECsLOB1, and Xcc pthA4 ::Tn5 containing individual dTALE constructs were infiltrated into fully expanded Valencia sweet orange leaves (OD 600 = 0.2). Representative results were chosen from three independent experiments. A. Schematic representation inoculation. B. dTALEOrange1.1t00600, C. dTALECs2g27100, D. dTALECs7g18460, E. dTALECs9g17380, F. dTALECs2g20750D, G. dTALECs7g32410, H. dTALCs6g17190. Discussion In this study, we have demonstrated that either dTALECs7g32410 (CsEXP2) or dTALECs2g20750 (CsEG1) can cause water soaking and pustule symptoms, which are common phenomena during Xcc infection of citrus leaves [ 2 ]. dTALECs7g18460 and dTALECs6g17190 do not cause water soaking and pustules, but cause slightly yellowing. The canker like symptoms caused by either dTALECs7g32410 or dTALECs2g20750 individually are much less than the combined effect of 5 dTALEs (Cs2g20750, Cs6g17190, Cs7g32410, Cs7g18460, Orange1.1t00600) and 7 dTALEs (5 dTALEs plus dTALECs2g27100 and dTALECs9g17380), indicating a coordinated effect among CsEXP2 and CsEG1 or other 5 direct targets of CsLOB1 play incremental roles in canker symptom development. CsEXP2 (Cs7g32410) encodes an expansin protein whereas CsEG1 (Cs2g20750) encodes an endoglucanase protein. Expansins are non-enzymatic cell wall-loosening proteins. Expansins can disrupt non-covalent bonding between the cellulose microfibrils and cell wall matrix polysaccharides, which leads to extension of cell wall and cell expansion [ 39 ]. In contrast, endoglucanases enzymatically cleave β-1,4-glycosidic bonds such as those found in cellulose and xyloglucan [ 40 ]. Endoglucanases also play important roles in cell expansion. Xcc is known to cause enlarged cells (hypertrophy), a kind of cell expansion. Thus, we have provided direct evidence that CsEXP2 (Cs7g32410) and CsEG1 (Cs2g20750) are direct targets of CsLOB1 involved in canker symptom development. It is noteworthy that Cs2g20750 was identified to be a direct target of CsLOB1 via Chip-seq elsewhere [ 20 ]. Separation of cellulose microfibrils during cell expansion causes cell water uptake, thus triggering water soaking. Cs7g18460 encodes a glucomannan 4-beta-mannosyltransferase 11 whereas Cs6g17190 encodes a gibberellin-regulated protein 5. Glucomannan 4-beta-mannosyltransferases are involved in synthesis of galactomannan, a non-cellulosic polysaccharides of plant cell wall [ 41 ]. Gibberellin-regulated proteins have been reported to be involved in multiple plant growth and development processes [ 42 ]. How Cs7g18460 and Cs6g17190 contribute to canker symptom development remains to be explored. Neither individual dTALEs nor mixed dTALEs can completely mimic the citrus canker symptoms caused by wild type Xcc in terms of the magnitude of water soaking, and pustule formation. Specifically, cell death was observed in the late stage of Xcc infection [ 43 ], but not in inoculation with dTALEs. This suggests other genes in addition to the 7 CsLOB1 direct targets are involved in citrus canker symptom development. For instance CsLIEXP1 is a direct target of CsLOB1 and encodes another expansin gene [ 31 ]. It is probable that activation of CsLIEXP1 contributes to hypertrophy phenomenon induced by Xcc. Multiple cell wall enzyme genes were reported to be direct targets of CsLOB1 including orange1.1t02719 (pectin esterase) and CsCEL20 (cellulase) [ 20 , 33 ]. Pectate lyases, polygalacturonases, xyloglucan endotransglycosylase/hydrolase and other plant cell wall enzymes are involved in cell expansion [ 40 ]. Many genes encoding plant cell wall enzymes are positively regulated by CsLOB1 [ 20 , 25 , 34 ], thus contributing to hypertrophy and water soaking symptoms. Cs2g20600, which encodes a zinc finger C3HC4-type RING finger protein, is a direct target of CsLOB1 [ 30 ]. C3HC4-type RING finger proteins are E3 ubiquitin ligases, which might be involved in proteasomal degradation during cell death [ 44 ]. CsNCED1-1 is a direct target of CsLOB1 and is a critical enzyme in ABA biosynthesis. ABA is a plant hormone that plays a crucial role in leaf senescence [ 45 ]. Activation of CsNCED1-1 might contribute to the leaf drops caused by Xcc infection [ 43 ]. Interestingly dTALECsLOB1 causes very similar canker symptoms as wild type Xcc including water soaking, hypertrophy and hyperplasia except cell death, suggesting other factors induced by PthA4 might be responsible for the cell death development. In sum, we have identified 7 direct targets of CsLOB1 and shown that Xcc indirectly activates CsEXP2 and CsEG1 via PthA4-CsLOB1 to cause canker symptoms. Identification of direct targets of CsLOB1 provides alternative target genes for disease resistance improvement via genome editing. Materials and methods Bacterial strains, plasmids, plant materials, and DNA manipulation The bacterial strains and plasmids used in this study are listed in Supplemental Table 2 . E . coli cells were cultured in Luria Bertani broth (LB) medium at 37°C. Agrobacterium strains were grown in LB containing rifampicin at 28°C. All Xanthomonas citri pv . citri ( Xcc) mutant strains used in this study were derivatives of Xcc 306. Xcc strains were cultured in nutrient broth (NB) medium at 200 RPM or on nutrient agar (NA) plates at 28°C. When required, culture media were supplemented with appropriate antibiotics (kanamycin, 50 μg/mL; gentamicin, 10 μg/mL; spectinomycin, 100 μg/mL; rifampicin 50 μg/mL; and ampicillin, 100 μg/mL). Nicotiana benthamiana plants were cultivated in a growth chamber at 25°C with a 16 h light/8 h dark photoperiod. Four-to eight-week-old N. benthamiana plants were used for all the experiments. Citrus plants used in this study was Valencia sweet orange ( Citrus sinensis ). Plants were grown in a temperature-controlled (28°C) greenhouse under natural light conditions. View this table: View inline View popup Supplementary Table 2. Strains or plasmids used in this study Generation of GUS reporter and CsLOB1 transient expression constructs The binary vector pCAMBIA1380 was used to clone and express CsLOB1 in N. benthamiana . pCAMBIA1380 contains the CaMV 35S promoter and a HA-tag downstream of the multiple cloning site, and the CsLOB1 gene was cloned into the vector utilizing the Xba I and Sal I restriction sites. pCAMBIA construct was introduced into Agrobacterium strain EHA105 by freeze-thaw method [ 46 ]. The GUS reporter system was used to assess the transcriptional activation of select target genes by LOB1. Promoter sequences located ∼1 kb upstream of the translational start site in Cs1g16560, Cs7g32410, Cs6g17190, Cs2g27100, Cs9g17380, Cs2g27090, Cs2g21440, Cs7g18460, Cs5g33680, orange1.1t00600, Cs2g05560, Cs2g20750, and Cs2g20600 were cloned into the binary GUS reporter construct pCAMBIA1380 35S-GUS using primers listed in Supplemental Table 3 . Promoter constructs were transformed into Agrobacterium strain EHA105. Agrobacterium transformants were cultured in LB medium overnight. Cultures were harvested by centrifugation (4000 g for 10 min), washed, and resuspended in infiltration buffer (10 mM MgCl2, 0.2 mM acetosyringone and 200 mM MES, pH 5.6) to a final OD 600 =0.5. Buffer-supplemented Agrobacterium strains were incubated at room temperature for 2-4 h, then the CsLOB1 and reporter constructs were co-infiltrated (OD 600 = 1.0) into leaves of 4-7 weeks old N. benthamiana with needleless syringes for transient expression assays [ 31 ]. In quantitative assays, three leaf discs (1 cm) were collected at 2 dpi, and GUS activity was measured using 4-methylumbelliferyl-β-glucuronide described previously [ 47 ]. Proteins were quantified using the Bradford method. The experiment was repeated three times with three biological replicates for each treatment. View this table: View inline View popup Supplementary Table 3. Primers used in this study Construction of dTALEs dTALEs were assembled using “Golden Gate TALEN and TAL Effector Kit 2.0” as previously described [ 48 ] and cloned into pTAL2 as a final destination vector. A library of four basic repeats encoding RVDs NG, NI, HD, and NH, which correspond to target nucleotides T, A, C, and G, respectively, were used. The repeat regions of artificial dTALEs were assembled using the RVDs corresponding to the targeted nucleotides near the TATA box in the promoter regions of Cs7g32410, Cs6g17190, Cs2g27100, Cs9g17380, Cs7g18460, orange1.1t00600, Cs2g20750, Orange1.1t00817, Cs5g34710, and Cs5g06600 ( Fig. 2A ). The broad host destination vector pBBRNPth with the −23 to +444 N-terminal coding fragment of pthA4 and an HA tag cloned into pBBR1MCS-5 [ 43 ] was used for subcloning the dTALEs RVDs along with N- and C-terminal regions. The pTAL2 Pst I/ Eco RI fragments containing the dTALEs were cloned into pBBRNPth for expression in Xanthomonas , and dTALE sequences were validated by PCR, digestion, and sequencing. Detailed information on the dTALEs, including RVDs and targeted EBE sequences, is shown in Fig.2A . All constructs were introduced into Xcc pthA4 ::Tn5 by electroporation. Detection and confirmation of dTALE through western blot analysis The expression of dTALEs was confirmed by western blot using mouse anti-HA antibody. Xcc pthA4 mutant strains containing dTALEs constructs were killed in sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (100 mM Tris-HCl, pH 6.8; 9% β-mercapto ethanol, 40% glycerol, 0.0005% bromophenol blue, 4% SDS) via heating for 10 min at 95°C, which were subjected to gel electrophoresis afterwards. Separated proteins were transferred onto the nitrocellulose membrane (Biorad, USA). Proteins were detected by an anti-HA antibody (Sigma Aldrich, USA), and developed on the X-ray film using kit from (Thermo Scientific, USA) dTALEs delivery into plant cells via Xanthomonas dTALEs were delivered into plant cells via XccpthA4 ::Tn5. The pBBRNpth-dTALEs plasmids ( Supplementary Table 2 ), containing an HA-tag epitope in the c-terminus of dTALEs, were electroporated (2.5 kv, 5 ms) into X cc pthA4 ::Tn5 strain. The Xcc strains - WT, pthA4 ::Tn5 mutant and mutants containing dTALEs for targeting the gene of interest and, mutant containing dTALE for targeting CsLOB1 were cultured overnight in NA liquid medium with respective antibiotics. The bacterial cells were collected by centrifugation (5000 g, 10 min), washed twice with 10 mM MgCl 2 and, resuspended in 10 mM MgCl 2 to OD 600 = 0.2. The suspensions were infiltrated into fully expanded young leaves of Valencia sweet orange with needleless syringes. Infiltration of simply 10 mM buffer was used as a mock. The leaf phenotype was photographed at 14-21 dpi. These experiments were repeated three times with similar results. Plant inoculations, and quantification of bacterial populations Bacterial suspensions (OD 600 =0.2) for monitoring symptom development, RNA isolation and physiological measurements, were syringe-infiltrated into fully expanded young leaves of two- to four-year-old Valencia sweet orange plants. After inoculations, plants were kept in a temperature-controlled (28°C) greenhouse under natural light conditions. RNA isolation and reverse transcription-quantitative PCR (RT-qPCR) RNA was isolated from leaf tissues inoculated with Xcc WT, pthA4 ::Tn5 mutant, pthA4 ::Tn5 (dTALEpthA4), and pthA4 ::Tn5 carrying dTALEs targeting different genes. For each sample, three 1-cm-diameter leaf disks from inoculated areas were pooled from three leaves belonging to the same plant and frozen in liquid nitrogen immediately. Samples were ground into fine powder using TissueLyser II (QIAGEN, Hilden, Germany). Total RNA was extracted using RNeasy plant RNA isolation kit (Thermo Fisher Scientific, Waltham, MA, USA). For RT-qPCR, 1 µg RNA samples were treated with Qiagen DNA out solution and reverse-transcribed using a qScript cDNA Synthesis Kit (Qiagen, Germany). cDNAs were amplified with gene-specific primer ( Supplementary Table 3 ) using SYBR Green master mix (Thermo Fisher Scientific, USA) by the Quant Studio 3 Real-Time PCR System (Applied Biosystems Inc., Foster City, CA). The GAPDH gene was used as an endogenous control for normalization, and gene expression level was calculated by the comparative Ct method [ 49 ]. Competing Interests The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to N. Wang. Acknowledgments We thank Wang lab members for constructive suggestions and insightful discussions. 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Share The TALE effector PthA4 of Xanthomonas citri subsp. citri indirectly activates an expansin gene CsEXP2 and an endoglucanase CsEG1 via CsLOB1 to cause citrus canker symptoms Rikky Rai , Nian Wang bioRxiv 2025.03.20.644280; doi: https://doi.org/10.1101/2025.03.20.644280 Share This Article: Copy Citation Tools The TALE effector PthA4 of Xanthomonas citri subsp. citri indirectly activates an expansin gene CsEXP2 and an endoglucanase CsEG1 via CsLOB1 to cause citrus canker symptoms Rikky Rai , Nian Wang bioRxiv 2025.03.20.644280; doi: https://doi.org/10.1101/2025.03.20.644280 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7616) Biochemistry (17625) Bioengineering (13852) Bioinformatics (41825) Biophysics (21397) Cancer Biology (18524) Cell Biology (25417) Clinical Trials (138) Developmental Biology (13350) Ecology (19858) Epidemiology (2067) Evolutionary Biology (24277) Genetics (15581) Genomics (22459) Immunology (17698) Microbiology (40278) Molecular Biology (17134) Neuroscience (88400) Paleontology (666) Pathology (2823) Pharmacology and Toxicology (4812) Physiology (7632) Plant Biology (15106) Scientific Communication and Education (2042) Synthetic Biology (4281) Systems Biology (9807) Zoology (2266)