Characterization of Xanthomonas oryzae- Responsive cis-acting Element in the Promoter of Rice Race-Specific Susceptibility Gene Xa13

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1 Moleculr Plnt Volume 4 Number 2 Pges Mrch 2011 RESEARCH ARTICLE Chrcteriztion of Xnthomons oryze- Responsive cis-acting Element in the Promoter of Rice Rce-Specific Susceptibility Gene X13 Ting Yun, Xinghu Li, Jinghu Xio nd Shiping Wng 1 Ntionl Key Lbortory of Crop Genetic Improvement, Ntionl Center of Plnt Gene Reserch (Wuhn), Huzhong Agriculturl University, Wuhn , Chin ABSTRACT The rice X13 gene, whose promoter hrbors UPT (up-regulted by trnscription ctivtor-like [TAL] effector) box, UPT PthXo1, plys pivotl role in the rce-specific pthogenicity cused by Xnthomons oryze pv. oryze (Xoo) strin PXO99. PXO99 cuses rice disese by inducing X13. It is unknown, however, whether the UPT PthXo1 box is the only PXO99-responsive cis-regulting elements in the ctivtion of X13 expression. We nlyzed the expression of series of end- nd site-truncted nd site-mutted X13 promoters in rice nd the binding of PXO99 protein to the intct, prtil, or site-mutted UPT PthXo1 boxes. In the X13 promoter, UPT PthXo1 box is the only Xoo-responsive cis-cting element tht results in PXO99-induced X13 expression. The 5 -terminl second, third, nd fourth nucleotides of the box re importnt for bcteril protein binding nd gene ctivtion; muttion of ny one of these sites bolished PXO99-induced gene expression. Furthermore, the 3 -hlf of the UPT PthXo1 box is lso required for protein binding nd gene ctivtion. These findings will enhnce our understnding of the moleculr mechnism of the interction of rice nd Xoo vi UPT boxes nd TAL effectors. Key words: Bcteril blight; disese; Oryz stiv; UPT box; Xnthomons oryze. INTRODUCTION Bcteri blight, which is cused by Xnthomons oryze pv. oryze (Xoo), is one of the most devstting diseses restricting rice production. More thn 30 disese resistnce (R) genes tht medite rce-specific resistnce to Xoo hve been identified, nd six of them hve been chrcterized (Chu nd Wng, 2007). The recessive x13 is new type of R gene tht confers resistnce to Philippine Xoo strin PXO99 (Chu et l., 2006). PXO99 infection does not influence the expression of recessive x13, but it induces the expression of its dominnt (susceptible) llele X13; suppressing the expression of X13 cn result in the sme level of resistnce to PXO99 s conferred by x13 in rice (Chu et l., 2006). Sequence nlysis of series of rice lines crrying either dominnt X13 or recessive x13 reveled set of recessive lleles of x13, whose encoding proteins re different from or identicl to tht encoded by dominnt X13. However, ll the recessive lleles hd nucleotide substitutions, deletions, or insertions in promoter region corresponding to the 86 to 69 region of the promoter of dominnt X13, suggesting tht promoter muttions my result in x13-medited disese resistnce (Chu et l., 2006). Promoter swp nlyses further confirmed tht the expressionl non-rection to PXO99 infection cused by promoter muttion, not its protein composition, is the key fctor for x13-medited resistnce (Yun et l., 2009). Thus, the dominnt X13 is rce-specific susceptibility gene. Activtion of X13 is required for the development of disese cused by PXO99. A recent study hs reveled tht PXO99 is sensitive to copper (Yun et l., 2010), which is n essentil micronutrient of plnts nd n importnt element for number of pesticides in griculture. PXO99 overcomes rice defense by regulting X13, 1 To whom correspondence should be ddressed. E-mil swng@mil. hzu.edu.cn, fx , tel ª The Author Published by the Moleculr Plnt Shnghi Editoril Office in ssocition with Oxford University Press on behlf of CSPP nd IPPE, SIBS, CAS. This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution Non-Commercil License ( org/licenses/by-nc/2.5), which permits unrestricted non-commercil use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. doi: /mp/ssq076, Advnce Access publiction 5 Jnury 2011 Received 19 September 2010; ccepted 18 November 2010

2 Yun et l. d Rice Pthogen-Responsive cis-element 301 which incorportes with nother two genes to remove copper from the xylem vessels, where Xoo multiplies nd spreds to cuse disese. The dominnt X13 is lso known s Os8N3, whose expression cn be induced by trnscription ctivtor-like (TAL) effector PthXo1 injected into rice through the type III secretion system of Xoo strin PXO99 (Yng et l., 2006). Effectors secreted by pthogenic bcteri ply n essentil role in promoting diseses in plnts (Ky nd Bons, 2009). Severl studies hve demonstrted tht pthogen TAL effectors cn trnscriptionlly ctivte host genes by directly intercting with the cis-regulting elements, nmed UPT (up-regulted by TAL effectors) boxes, in the promoters of corresponding host susceptibility genes to promote diseses or in R genes to induce defense responses (Ky et l., 2007, 2009; Römer et l., 2007, 2009, 2009b). This interction is determined by the specific piring of the repet-vrible diresidues (RVDs) of the repet domin of TAL effector nd the nucleotides of UPT box, with one RVD piring to one specific nucleotide in the UPT box (Boch et l., 2009; Moscou nd Bogdnove, 2009). Thus, bsed on the piring codes of given TAL effector, one cn predict puttive UPT box tht my interct with this TAL effector. A recent study reveled tht the TAL effector PthXo1 from Xoo strin PXO99 directly binds to UPT box, UPT PthXo1 (consisting of 25 nucleotides), in the X13 promoter to medite gene ctivtion (Römer et l., 2010). However, it is unknown whether the UPT PthXo1 box is the only cis-regulting element tht is responsible for Xoo-induced ctivtion of X13. In the present study, we nlyzed the expression of truncted nd mutted X13 promoters in rice. We lso exmined the binding of PXO99 totl proteins to the intct, incomplete, or site-mutted UPT PthXo1 box nd corresponding DNA frgments from recessive x13 lleles. Our results suggest tht, in the X13 promoter, UPT PthXo1 box is the only cis-cting element tht results in PXO99-induced X13 expression. The importnt nucleotide sites of the UPT PthXo1 box nd the neighboring sites of this box needed for efficient ctivtion of the gene re discussed. RESULTS Identifiction of the Pthogen-Responsive Region of the X13 Promoter Sequence nlysis suggested tht the puttive TATA boxes, which provide the binding sites for RNA polymerse, were t 34 for the promoters of both the dominnt X13 gene (P X13 ) nd recessive x13 gene (P x13 ) (Figure 1A). Bsed on the puttive loctions of TATA boxes, previous identifiction of the promoter regions of X13 nd x13 genes (Yun et l., 2009), nd the loction of UPT box for PthXo1 effector binding (Römer et l., 2010), the DNA frgment locted t the 1418 to 6 region of X13 nd the corresponding DNA frgment locted t the 1615 to 6 region of x13 were A -34 (TATA) -34 (TATA) P X (ATG) (ATG) P 252 x13 X13 x13-80 (UPT PthXo1 ) Corresponding to -80 of P X B P X P x P X P x P X P x P X P X P X13-R50 substitution deletion insertion Figure 1. The Structures of Promoters P X13 nd P x13 from IR24 (Crrying X13) nd IRBB13 (Crrying x13), Respectively, nd Truncted Promoters. IR24 nd IRBB13 re ner-isogenic rice lines. (A) The predicted bsl regultory element TATA in P X13 nd P x13. The trnscription initition site is indicted s +1. The nucleotide substitution, deletion, or insertion in P x13 compred with P X13 were bsed on previous report (Chu et l., 2006); the figure with sign of deletion or insertion indictes the numbers of nucleotides inserted or deleted nd the sign without figure represents singlenucleotide deletion or insertion. ATG, trnsltion strt codon. (B) The full-length nd truncted promoters. fused with the reporter gene b-glucuronidse () to detect the pthogen-responsive regions; two truncted promoters P X nd P X for P X13 nd P x nd P x for P x13 were lso fused with (Figure 1B). These constructs were trnsferred seprtely into rice, nd ech construct generted pproximtely 20 independent positive trnsgenic plnts. Ech trnsgenic plnt ws divided into two prts by seprting the tillers t the tillering stge: one prt for inocultion with Xoo strin PXO99 nd nother prt for mock-inocultion t the booting (pnicle development) stge. Becuse X13 expression ws mrkedly induced t 1 5 d fter PXO99 infection (Chu et l., 2006; Yun et l., 2009, 2010), the expression level of mrker gene in the trnsgenic plnts ws exmine from 8 h to 5 d fter infection. The expression of in the trnsgenic plnts crrying P X13 : ws strongly induced t 2 d nd incresed continully until 5 d fter PXO99 infection compred with mock-inoculted control plnts (Figure 2). Neither PXO99 infection nor mock-inocultion influenced expression in the trnsgenic plnts crrying P x13 : (Figure 2). The level in the plnts crrying P X13 :- t 5 d fter PXO99 infection ws 23-fold higher thn tht in the sme plnts t 5 d fter mock-inocultion. Plnts crrying P X nd those crrying P X showed similr induced expression pttern of to plnts crrying P X13 : fter PXO99 infection (Figure 2). However, plnts crrying P X nd those crrying P x showed significntly higher level (P, 0.01) of thn the plnts crrying P X13 : or P x13 : t 8 h fter infection or even without

3 302 Yun et l. d Rice Pthogen-Responsive cis-element pthogen infection, respectively. Compred with mockinocultion, PXO99 infection induced pproximtely two nd three-fold increses of levels in plnts crrying P X t 8 h, 2, nd 5 d fter infection, respectively. PXO99 infection did not mrkedly influence expression in plnts crrying P x compred with the sme plnts fter mock-inocultion. Plnts crrying P X nd those crrying P x showed similr levels of to the plnts crrying P X13 : or P x13 : when without pthogen infection. PXO99 infection showed pproximtely nine- nd 10-fold increses in levels in plnts crrying P X s compred with the sme plnts fter mock-inocultion t 2 nd 5 d fter infection, respectively (Figure 2). PXO99 infection did not mrkedly influence levels in the plnts crrying P x compred to the sme plnts fter mockinocultion. These results suggest the following possibilities. First, the 1418 to 935 region of P X13 nd the corresponding 1615 to 1148 region of P x13 my contin PXO99- independent cis-regulting element(s) tht suppress gene expression. Second, the 934 to 395 region of P X13 nd the corresponding 1147 to 609 region of P x13 my contin PXO99-independent cis-regulting element(s) tht stimulte pmol Mu/min/mg protein pmol Mu/min/mg protein Time fter PXO99 inocultion (h) P X13 P X P X P x13 P x P x Time fter mock inocultion (h) Figure 2. The Expression of Driven by Ntive (P X13 nd P x13 ) or Truncted (P X13-934, P X13-394, P x , nd P x ) Promoters fter PXO99-Inocultion or Mock-Inocultion t the Booting Stge. ctivity ws determined by mesuring the mount of 4- methylumbelliferone (Mu) produced under the ctlysis of in 1 mg totl protein per minute. Sixteen to 22 T 0 trnsgenic plnts crrying ech construct were used for nlyses. For ech time point exmined, lef frgments were collected from ll the plnts crrying the sme construct nd mixed to prepre smple. The ctivity t ech time point ws the verge of three mesurements 6 stndrd devition. The indictes tht significnt difference (P, 0.01) ws detected between pthogen-inoculted nd mockinoculted plnts in the sme time point. gene expression. Lst, only the 394 to 6 region of P X13 my contin PXO99-responsive element(s) tht induce gene expression. The 394 to 6 region of P X13 hrbors PXO99-responsive element, the UPT PthXo1 box, locted t 80 to 56 (Figure 1A; Römer et l., 2010). To determine whether this region my contin other PXO99-responsive elements in ddition to the UPT box, two dditionl 5 -end truncted promoters, P X nd P X13-66, nd one 3 -end truncted promoter, P X13-R50,ofP X13 were fused with (Figure 1B). The three constructs s well s the construct crrying P X13 : were trnsiently expressed in rice clli. The expression of in the clli crrying P X : ws strongly induced by PXO99-inocultion compred with mock-inoculted control clli, s ws the cse in the clli crrying P X13 : (Figure 3). PXO99-inocultion did not obviously influence expression in the clli crrying P X13-66 : or P X13-R50 : compred with mock-inoculted clli. Becuse the P X13-66 : construct only hrbored incomplete UPT PthXo1 box, these results suggest tht the 121 to 65 region of P X13 my contin nother PXO99-responsive element tht induces gene expression or the 121 to 6 region my contin only one PXO99-responsive element, the UPT PthXo1 box. However, PXO99 infection did not obviously influenced expression in the clli crrying P X13-R50 :, which hrbored the 121 to 50 region; this my be due to the lck of the bsl trnscriptionl element, the TATA box. This ssumption is pmol Mu/min/mg protein pmol Mu/min/mg protein ck P X13 Time fter PXO99 inocultion (h) P X13-66 P X P X13-72 ck Time fter mock inocultion (h) Figure 3. Trnsient Expression of Driven by Ntive (P X13 ) nd Truncted (P X13-121, P X13-66, nd P X13-R50 ) Promoters in Rice Clli. ctivity ws determined by mesuring the mount of 4- methylumbelliferone (Mu) produced under the ctlysis of in 1 mg totl protein per minute. Ech smple ws from 4.5 ml clli; ll the clli were mixed to prepre smple. The ctivity of ech smple ws the verge of three mesurements 6 stndrd devition. ck, without pthogen- or mock-inocultion. The indictes tht significnt difference (P, 0.01) ws detected between pthogen-inoculted nd mock-inoculted plnts in the sme time point. This experiment ws biologiclly repeted twice nd similr results were obtined.

4 Yun et l. d Rice Pthogen-Responsive cis-element 303 supported by evidence tht the levels in the clli crrying P X13-66 : were constntly pproximtely three-fold higher thn those in the clli crrying P X13-R50 : with either PXO99- or mock-inocultion. More Xoo Proteins Bind to the Promoter Frgment of X13 thn to the Promoter Frgment of x13 Although our findings indicted tht the 121 to 66 region my hrbor PXO99-responsive element, previous study showed tht region corresponding to the 86 to 69 region of P X13 might be responsible for PXO99-induced expression of dominnt X13, bsed on the comprison of the promoter sequences of seven rice lines crrying dominnt X13 nd 11 rice lines crrying recessive x13 or its recessive lleles (Chu et l., 2006). To determine whether the protein(s) of PXO99 could directly bind to the puttive PXO99-responsive element of P X13, 14- to 15-nt DNA probes from the 86 to 69 region of P X13, which hrbored the 5 -hlf of the UPT PthXo1 box, nd the corresponding regions of some promoters of recessive x13 nd its recessive lleles were designed bsed on sequence-specific nlysis nd used for protein binding nlyses (Figure 4A). Becuse the TAL effector PthXo1 of PXO99 trnscriptionlly ctivtes X13 (Yng et l., 2006), we expected tht PXO99 proteins would bind more intensively to the probe from P X13 thn the probes from P x13, if there ws protein binding. Unexpectedly, n electrophoretic mobility shift ssy (EMSA) showed tht PXO99 proteins bound more intensely to the four probes, R15-Tep 1, R15-IRBB13, R14-AUS274, nd R15-AC19-1 1, from the promoters of recessive x13 nd its recessive lleles thn to probe D15-IR24 from P X13 (Figure 4B). The protein-binding of D15-IR24 nd R15-Tep 1 ws reduced or bolished by the competition of unlbelled probes, indicting specificity of the binding. To determine which nucleotide influenced the binding of PXO99 proteins, three site-mutted short DNA probes (D15M1, D15M2, nd D15M3) were used for nlysis of protein binding (Figure 4A). By compring the protein binding intensity of D15M1, D15M2, nd D15M3 with tht of D15-IR24, substitution of C with A or A with G in probes D15M2 nd D15M3, respectively, ppered to be importnt for protein binding (Figure 4B). A recent study reported tht the UPT PthXo1 box of P X13 consists of 25 nucleotides (Figure 4A; Römer et l., 2010). Thus, we designed new set of long DNA probes tht hrbored the full-length UPT PthXo1 box from X13 or the corresponding regions from x13 or its recessive lleles (Figure 4A). Among these long probes, R28-IRBB13, R28-Tep 1, R28-AUS274, nd R28-AC from the promoters of recessive x13 (IRBB13) nd its recessive lleles (Tep 1, AUS274, nd AC19-1 1), respectively, hrbored DNA frgments tht hd 1-, 3-, 10-, or 16-nucleotide differences from the UPT PthXo1 box. EMSA showed tht PXO99 proteins bound intensively to probe D28-IR24 hrboring the UPT PthXo1 box, but only very wekly to probes D28-IRBB13, D28-Tep 1, D28-AUS274, nd D28-AC (Figure 4C). Substitution of the third C nucleotide of the UPT PthXo1 box with A (probe D28M2) or the fourth A nucleotide with G (D28M3) mrkedly reduced the binding (Figure 4C). However, substitution of the sixth C of the UPT PthXo1 box with A (probe D28M1) did not influence the binding ffinity of PXO99 proteins. The protein-binding signls of the probes were bolished by the competition of unlbelled probes, indicting specificity of the binding (Figure 4C). These results suggest tht n intct UPT PthXo1 box is essentil for bcteril protein binding. Furthermore, t lest the second, third, nd fourth nucleotides of the box re importnt for bcteril protein binding, wheres the sixth nucleotide of this box my not be importnt for protein binding. Muttion of UPT PthXo1 Box Influences PXO99-Induced Trnscriptionl Activity To scertin whether the differentil binding ctivities of the vrious probes to bcteril proteins ffected trnscriptionl regultion of the promoter, we constructed four site-mutted promoters (P X13D79T, P X13D78A, P X13D77G, nd P X13D75A ) of P X13 hrboring the UPT PthXo1 box, in which the second, third, fourth, nd sixth nucleotides were mutted, respectively (Figure 5A). These mutted promoters were fused with nd trnsferred seprtely into rice. Ech construct generted pproximtely 20 independent positive trnsgenic plnts. Ech plnt ws divided into two prts by seprting the tillers t the tillering stge for inocultion with Xoo strin PXO99 nd mock-inocultion, respectively. Trnsgenic plnts crrying different promoter constructs showed the similr levels of expression when mock-inoculted. The PXO99-induced expression of in the trnsgenic plnts crrying P X13D79T, P X13D78A,orP X13D77G ws completely or prtilly suppressed compred to the expression of in the plnts crrying P X13 : (Figure 5B). However, plnts crrying P X13D75A showed similr level of PXO99-induced expression to the plnts crrying P X13 :. The mutted UPT PthXo1 boxes in the promoters P X13D79T, P X13D78A, nd P X13D77G corresponded to DNA probes R28-Tep1, D28M2, nd D28M3, respectively, which showed only wek binding of PXO99 proteins (Figure 4C). The mutted UPT PthXo1 box in P X13D75A ws consistent with probe D28M1, which showed strong binding to PXO99 proteins (Figure 4C). These results suggest tht the suppression or loss of PXO99-induced trnscriptionl ctivtion in the trnsgenic plnts crrying P X13D79T, P X13D78A,or P X13D77G my hve resulted from the unvilbility or low ffinity of binding bcteril protein to the promoters due to the muttion of the key sites of the UPT PthXo1 box. These results lso suggest tht the UPT PthXo1 box is the only PXO99- responsive element in the promoter of X13. We lso constructed seven site-truncted promoters. Three to 11 nucleotides either in front of the UPT PthXo1 box (P X13-F3 ) or t 3 -terminl of the UPT PthXo1 box (P X13-E3, P X13-E5, P X13-E7, P X13-E9, P X13-E11, nd P X13-E13 ) were deleted from P X13 promoter (Figure 6A). These site-truncted constructs nd the construct crrying P X13 : were trnsiently expressed in seprte rice clli. The expression of in the clli crrying

5 304 Yun et l. d Rice Pthogen-Responsive cis-element Figure 4. The Binding Ability of the Promoter Probes of Dominnt X13 nd Recessive x13 to the Totl Proteins of Xoo Strin PXO99 Anlyzed by EMSA. The binding ssys were biologiclly repeted twice, with similr results ((B) nd (C)). (A) Probe sequences. Rice line IR24 crries dominnt X13. Rice lines IRBB13, Tep 1, AUS274, nd AC crry different lleles of recessive x13; nother five rice lines, Chinsurh Boro2 (11484), Chinsurh Boro2 (11760), Chinsurh Boro2 (50930), Long Grin (64950), nd BJ1 tht crry different x13 lleles from tht of Tep 1, hve the sme DNA sequence s Tep 1 in the region corresponding to the 86 to 69 region of P X13 (Chu et l., 2006). Probe D28-IR24 hrbors the UPT PthXo1 box of P X13 (bold itlic letters). Compred with D28-IR24, the substitution sites of the probes from the promoters of x13 nd its recessive lleles re underlined. Muttion sites re shown in lower-cse letters. (B) The binding bility of PXO99 totl proteins to different short (14- or 15- nt) probes. The lbeled probes plus 50- or 100-fold unlbelled competitor probes or without plus competitor (0) were used for the binding ssys. ck, without PXO99 proteins; +, with PXO99 proteins. (C) The binding bility of PXO99 totl proteins to different long (28-nt) probes. The lbeled probes plus 50- fold unlbelled competitor (C) probes or without plus competitor (0) were used for the binding ssys. only P X13 : or P X13-F3 : but not other constructs ws significntly induced (P, 0.01) by PXO99-inocultion compred to mock-inocultion (Figure 6B). However, level in the clli crrying P X13-F3 : ws significntly lower thn tht in the clli crrying P X13 : t 4 24 h fter infection (Figure 6B). These results suggest tht both the flnking sequence nd the 3 -terminl prt of UPT PthXo1 box my be importnt for Xoo-induced expression. DISCUSSION Xoo-induced X13 expression is criticl for X13-fcilited susceptibility (Yun et l., 2009, 2010). The PthXo1 effector of Xoo strin PXO99 trnscriptionlly ctivtes X13 by binding to cis-cting element, the UPT PthXo1 box, in the gene s promoter (Yng et l., 2006; Römer et l., 2010). We nlyzed series of end- nd site-truncted nd site-mutted promoters, nd our findings suggest tht the UPT PthXo1 box is the only PXO99- responsive element in the X13 promoter. Thus, interction of PthXo1 effector nd UPT PthXo1 box results in the susceptibility of rice to PXO99. The promoters of recessive R gene x13 nd its recessive lleles crry mutted UPT PthXo1 boxes, which is the key reson for x13-medited resistnce. Although the present study used the totl proteins of PXO99 insted of PthXo1 for DNA-protein binding nlyses, the proteins bound to the DNA probe hrboring the UPT PthXo1 box re likely minly the PthXo1 effector, s supported by the following evidence. First, EMSA showed tht PXO99 proteins bound intensively to the DNA probe hrboring the full-length UPT PthXo1 box but wekly to the probes hrboring mutted UPT PthXo1 boxes from the promoters of recessive x13 nd its recessive lleles (Figure 4C). These results gree with recent report tht His:PthXo1 fusion protein binds strongly to the DNA frgment hrboring the UPT PthXo1 box but to lesser extent to the corresponding promoter region of recessive x13 (Römer et l., 2010). Second, replcement of the second G nucleotide of the UPT PthXo1 box with T in nturl muttion (the recessive llele of x13 from rice vriety Tep 1) mrkedly

6 Yun et l. d Rice Pthogen-Responsive cis-element 305 Figure 5. Expression of Driven by Ntive (P X13 ) or Mutted (P X13D79T, P X13D75A, P X13D78A, nd P X13D77G ) Promoters fter PXO99 Infection or Mock-Inocultion t the Booting Stge. (A) The structures of mutted promoters nd the sites of muttions. The UPT PthXo1 box of P X13 is shown with bold itlic letters. The figure with sign of substitution indictes the site of nucleotides substitution. (B) Expression of in trnsgenic plnts. ctivity ws determined by mesuring the mount of 4- methylumbelliferone (Mu) produced under the ctlysis of in 1 mg totl protein per minute. Twenty to 36 T 0 trnsgenic plnts crrying ech construct were used for nlyses. For ech time point exmined, lef frgments were collected from ll the plnts crrying the sme construct nd mixed for prepring smple. The ctivity t ech time point ws the verge of three mesurements 6 stndrd devition. ck, without pthogenor mock-inocultion. The sterisk (*) indictes tht significnt difference (P, 0.01) ws detected between PXO99-inoculted plnts nd non-inoculted control (ck) plnts crrying the sme promoter. reduced protein binding (Figure 4C). Likewise, substitution of the second nucleotide of the UPT PthXo1 box with A, C, or T significntly reduced or lmost complete negted PthXo1- medited promoter ctivtion (Römer et l., 2010). Finlly, the sme muttions in the UPT PthXo1 box tht reduced PXO99 protein binding bolished PXO99-induced X13 expression (Figure 5), which ws similr to the knockout of PthXo1 in PXO99 (Yng et l., 2006). Recently, severl UPT boxes hve been predicted by using the TAL effector code, the RVDs tht re the hypervrible residues 12 nd 13 in ech repet unit of the repet domin of TAL effectors (Boch et l., 2009; Moscou nd Bogdnove, 2009). Although bout hlf of the identified RVDs of TAL effectors hve degenerted piring nucleotides bsed on the identified nd predicted UPT boxes, with some RVDs codes vrying between two to four nucleotides (Boch et l., 2009; Moscou nd Bogdnove, 2009), this degenercy ppers to be influenced by the position of RVD in TAL effectors or/nd by the position of nucleotide in UPT box. For exmple, the RVD NN ppers to recognize four types of nucleotide in UPT boxes (Boch et l., 2009; Moscou nd Bogdnove, 2009). However, mtching the NN-type RVD of PthXo1 to the 5 -end second G nucleotide but not the A, C, or T nucleotide of the UPT PthXo1 box is crucil for the interction of the PthXo1 nd UPT PthXo1 box (Römeretl.,2010). Our present results lso showed tht substitution of the second G to T in nturl muttion mrkedly reduced PXO99 protein binding nd bolished PXO99-induced gene expression. Furthermore, the 5 -end third nd forth nucleotides of the UPT PthXo1 box re lso importnt for PXO99 protein binding nd gene trnscriptionl ctivtion. Both the RVDs piring to the third nd sixth nucleotides re HD-type (Römer et l., 2010), which is suggested to hve degenerted piring nucleotides in the UPT boxes (Boch et l., 2009; Moscou nd Bogdnove, 2009). Interestingly, substitution of the third C to A in the UPT PthXo1 box bolished PXO99-induced gene expression nd substitution of the sixth C to A did not influence gene ctivtion (Figure 5). Similr results were observed in nother study. The RVDs of TAL effector AvrBs3 piring the 14th nd 15th nucleotides of the UPA AvrBs3 box (lso UPT box) in the promoter of pepper R gene Bs3 re HDtype, but substitution of the 15th nucleotide C to A in the UPA Bs3 box bolished the Bs3-medited hypersensitive response, while substitution of the 14th nucleotide C to A did not influence Bs3 function (Römeretl.,2009b).These results suggest tht the three-dimensionl positions of some RVDs my influence the interction of TAL effector nd UPT box. One RVD pirs with one nucleotide in UPT box in the host bcterium interction; thus, the number of RVDs in TAL

7 306 Yun et l. d Rice Pthogen-Responsive cis-element Figure 6. Trnsient Expression of Driven by Ntive (P X13 ) nd Site-Truncted (P X13-F3, P X13-E3, P X13-E5, P X13-E7, P X13-E9, P X13-E11, nd P X13-E13 ) Promoters in Rice Clli. (A) The structures of site-truncted promoters nd the sites of deletion. The UPT PthXo1 box of P X13 is shown with bold itlic letters. The figure with sign of deletion indictes the numbers of nucleotides deleted. (B) Expression of driven by different promoters in rice clli. ctivity ws determined by mesuring the mount of 4-methylumbelliferone (Mu) produced under the ctlysis of in 1 mg totl protein per minute. Ech smple ws from 4.5 ml clli; ll the clli were mixed to prepre smple. The ctivity of ech smple ws the verge of three mesurements 6 stndrd devition. One (*) or two (**) sterisks indicte tht significnt difference between plnts crrying P X13 : nd P X13-F3 : in the sme time point ws detected t P, 0.05 or P, 0.01, respectively. ck, without pthogen- or mock-inocultion. This experiment ws repeted twice biologiclly nd similr results were obtined. effector determines the size of the corresponding UPT box (Boch et l., 2009; Moscou nd Bogdnove, 2009). However, the length of functionl UPT box is not lwys s long s tht predicted using the TAL effector code. The nucleotides in the 3 -end of some UPT boxes pper to not be required for gene ctivtion. The AvrX27 effector contins 17 RVDs (Moscou nd Bogdnove, 2009). Omission of the lst three nucleotides of the predicted UPT AvrX27 box cn still trigger hypersensitive response by AvrX27 (Römer et l., 2009). The AvrBs3Drep16 effector contins 14 RVDs (Römer et l., 2010). Omission of the lst two nucleotides of the predicted UPA AvrBs3Drep16 box (lso UPT box) cn lso trigger hypersensitive response by AvrBs3Drep16 (Römer et l., 2009b). A recent study reported tht t lest 6.5 RVDs were required to recognize the trget DNA box nd to ctivte gene expression, nd 10.5 or more RVDs could efficiently ctivte gene expression (Boch et l., 2009). Our present results showed tht the terminl nucleotides of the UPT PthXo1 box could not provide correct pthogen protein binding (Figure 4). Even deletion of the three nucleotides t the 3 -end of the UPT PthXo1 box bolished PXO99-induced gene expression (Figure 6). Likewise, n incomplete UPA AvrBs3Drep16 box (lcking the first nucleotide) hd lower ffinity for the AvrBs3Drep16 effector thn the complete UPA AvrBs3 box (Römer et l., 2007). However, the complete UPA AvrBs3Drep16 box showed higher ffinity for AvrBs3Drep16 thn the complete UPA AvrBs3 box (Römer et l., 2009b). These results suggest tht in some cses, n incomplete UPT box my result in non-specific protein binding. The 5 -terminl Tof UPT boxes hs been reported to be crucil to trnscriptionl ctivtion by TAL effectors (Boch et l., 2009;

8 Yun et l. d Rice Pthogen-Responsive cis-element 307 Römer et l., 2009b, 2010). Interestingly, the flnking sequence of the UPT PthXo1 box ppers to influence PXO99-induced gene expression. The deletion of the three nucleotides flnking the 5 -end of the UPT PthXo1 significntly reduced the expression levelofthegenectivtedbypxo99(figure6). Theneighboring nucleotides of t lest some plnt cis-cting elements lso contributetohigh-ffinitybindingofregultingproteins(ciolkowski et l., 2008). It remins to be demonstrted whether it is common feture tht neighboring nucleotides of UPT boxes contributetobindingffinityoftaleffectors. Inconclusion, the moleculr mechnism of the specific interctions of TAL effectors nd UPT boxes still needs to be refined. Investigting the binding of TAL effectors to UPT boxes t the three-dimensionl level my help to clrify these issues. METHODS Constructing End-Truncted Promoters Truncted promoters of dominnt X13 gene (P X13 ) nd recessive x13 gene (P x13 ) were obtined by PCR mplifiction from the 5 -end using the intermedite vector contining P X13 nd P x13 (Yun et l., 2009), respectively, s templte. The 5 -endprimersofx13p-f(5 -GGATCCGATGTTGAGCTTTAGGAT- TAGCGGGTT-3 ), 5 -deletion-1147 (5 -GGATCCTCCCTTTCTT- CAAGTTACCTCTCTC-3 ), nd 5 -deletion-608 (5 -GGATCCGCAT CATTGTCCATGGTTGT-3 ), which were specific to both P X13 nd P x13 nd contined the BmHI digestion site (underlined), s well s primers XP-90F (5 -GGATCCGAAATATCAAGCACAAG- 3 ) nd XP-60F (5 -GGATCCCTGTACACCACCAAAAG-3 ), which were specific to P X13, were used in combintion with the 3 - end primer X13-promR (5 -GGCAAGCTTGGCCTTGGCCATGGCT- CAGT-3 ), which ws specific to both P X13 nd P x13 nd contined the HindIII digestion site (underlined). The 5 -end primer XP-90F ws lso used in combintion with the 3 -end primer XP-60R (5 -AAGCTTCTTTTGGTGGTGTACAG-3 ), which ws specific to P X13 nd contined the HindIII digestion site (underlined). The PCR products were ligted to vector pgem- T (Promeg Corportion, Mdison, WI, USA) for sequencing, then the truncted promoter frgments were digested with BmHI nd HindIII nd ligted to vector pcambia1381 to form truncted promoters fused with reporter gene. Rice Trnsformtion The promoter constructs were trnsferred into rice vriety Mudnjing 8 (Oryz stiv L. ssp. jponic) byagrobcteriummedited trnsformtion (Lin nd Zhng, 2005). A pir of PCR primers, GusF (5#-CCAGGCAGTTTTAACGATCAGTTCGC-3#) nd GusR (5#-GAGTGAAGATCCCTTTCTTGTTACC), designed ccording to the sequence of ws used for detecting positive trnsgenic plnts. Pthogen Inocultion Rice plnts were inoculted with Xoo strin PXO99 by the lefclipping method t the booting (pnicle development) stge (Sun et l., 2004). Mock-inoculted plnts were treted under the sme conditions except tht the Xoo suspension ws replced with wter. Anlysis of Activity Lef frgments bout 1 cm long right next to the inocultion sites were used for nlysis of expression. Quntittive nlyses of ctivity were conducted s described previously (Ci et l., 2007). Totl protein concentrtion in the superntnt ws quntified with the Brdford ssy (Brdford, 1976). protein in the superntnt ws determined fluorometriclly with n INFINITE 200 (Tecn Austri Gmbh, Ltd, Grodig, Austri). Agrobcterium-Medited Trnsient Expression The clli of rice vriety Zhonghu 11 (O. stiv ssp. jponic) were prepred s described previously (Lin nd Zhng, 2005). Agrobcteri contining different trnsformtion construct were co-cultured with the clli for over 16 h in MS medium. The clli were then wshed 10 times using utoclved wter nd dried in lminr flow cbinet. The clli were treted with Xoo strin PXO99 t 10 9 cfu ml 1 or mock-treted with utoclved wter for certin period of time. Site-Directed Muttion nd Trunction The GeneTilor Site-Directed Mutgenesis System (Invitrogen Life Technologies, Crlsbd, CA, USA) ws used for sitedirected muttion of X13 promoter s described previously (Ci et l., 2007). The puc19 plsmid tht contined the ntive promoter (P X13 ) used s PCR templte ws methylted before use. The mutgenic primer pir, in which the regultory element ws mutted, ws used to mplify the promotercontining trget muttion. Primer pirs X13PM1F (5 -AAAG CAAAGGTTAGATATTCATCTCCCCCT-3 )/X13PM1R (5 -ATATC- TAACCTTTGCTTTTTTTTTTC-3 ), X13PM2F (5 -CAAAGGTTAGA TATGCATATCCCCCTACTG-3 )/X13PM2R (5 -ATGCATATCTAAC- CTTTGCTTTTTTTTTTC-3 ), X13PM4F (5 -AAGCAAAGGTTAGA- TATGAATCTCCCCCT-3 )/X13PM4R (5 -CATATCTAACCTTTGCTT TTTTTTTTC-3 ), nd X13PM5F (5 -AGCAAAGGTTAGATATGCG- TCTCCCCCTAC-3 )/X13PM5R (5 -GCATATCTAACCTTTGCTTTTT- TTTTTC-3 ) were used for site-directed muttion of P X13,in which the muttion points were underlined. To help select the mutted construct, the PCR product ws trnsferred into Escherichi coli strin DH5-T1, in which the methylted plsmid could not replicte. The site-directed trunction of X13 promoter ws performed by nested PCR mplifiction using the intermedite vector contining P X13 (Yun et l., 2009) s templte. First, the forwrd primer BX13(IR24)M17F (5 -TAGATATGCATCTCCCCC- TACAAAAGTGGAG-3 ), whichcontineddeletioninthetrget site, ws used in combintion with the reverse primer X13-promR to mplify the downstrem of the deletion site. Simultneously, the reverse primer BX13(IR24)M17R (5 -GGGGGAGATGCATATCTAACCTTTGCTTTT-3 ), which ws

9 308 Yun et l. d Rice Pthogen-Responsive cis-element overlpped with BX13(IR24)M17F nd lso contined the sme deletion s in BX13(IR24)M17F, ws pired with the forwrd primer X13P-F to mplify the upstrem of the deletion site. The mplifiction products from the two PCR rections were purified nd mixed nd used s the templte for the second round of PCR using primers X13P-F nd X13-promR. Another six pirs of primers, BX13(IR24)M15F (5 -TAGATATGCATCTCCCCC- CAAAAGTGGAGGG-3 )/BX13(IR24)M15R (5 -GGGAGATGCA- TATCTAACCTTTGCTTTTTT-3 ), BX13(IR24)M19F (5 -GATATGC ATCTCCCCCTACTCAAAAGTGGAG-3 )/BX13(IR24)M19R (5 -TA GGGGGAGATGCATATCTAACCTTTGCTT-3 ), BX13(IR24)M21F (5 -TATGCATCTCCCCCTACTGTCAAAAGTGGAG-3 )/BX13(IR24) M21R (5 -AGTAGGGGGAGATGCATATCTAACCTTTGC-3 ), BX13 (IR24)M23F (5 -TGCATCTCCCCCTACTGTACCAAAAGTGGAG-3 )/ BX13(IR24)M23R(5 -ACAGTAGGGGGAGATGCATATCTAACCTT T-3 ), BX13(IR24)M25F(5 -CATCTCCCCCTACTGTACACCAAAAG TGGAG-3 )/BX13(IR24)M25R (5 -GTACAGTAGGGGGAGATG- CATATCTAACCT-3 ), nd BX13(IR24)M-3F (5 -GAAAAAAAAAA GCAAAGGTTAGTGCATCTCCCC-3 )/BX13(IR24)M-3R (5 -AACC TTTGCTTTTTTTTTTCTTGTGCTTGA-3 ), were lso used to construct other site-directed truncted promoters in the sme wy s described bove. Electrophoretic Mobility Shift Assy To isolte the totl proteins of Xoo strin PXO99, the fresh bcteri were ground with liquid nitrogen nd suspended in buffer (10 mm Tris-HCl t ph 8.0, 1 mm DTT, 200 lm phenylmethnesulfonyl fluoride) nd homogenized completely. The mixture ws centrifuged t 4 C t 1000 g to collect the superntnt. The protein content in the nucler extrct nd in the totl proteins of Xoo strin PXO99 ws quntified using the Brdford ssy. EMSA ws pplied s described previously (Qiu et l., 2007). Promoter Sequence Anlysis The TATA boxes of promoters P X13 nd P x13 were predicted using the computer progrms TSSP provided t the Softberry website ( nd PROSCAN ( bims.dcrt.nih.gov/molbio/proscn). Sttisticl Anlysis The significnt differences between control nd tretment of the smples were nlyzed by the pir-wise t-test instlled in the Microsoft Office Excel progrm. 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