Skip to main content

Identification of a virulence tal gene in the cotton pathogen, Xanthomonas citri pv. malvacearum strain Xss-V2–18



Bacterial blight of cotton (BBC), which is caused by the bacterium Xanthomonas citri pv. malvacearum (Xcm), is a destructive disease in cotton. Transcription activator-like effectors (TALEs), encoded by tal-genes, play critical roles in the pathogenesis of xanthomonads. Characterized strains of cotton pathogenic Xcm harbor 8–12 different tal genes and only one of them is functionally decoded. Further identification of novel tal genes in Xcm strains with virulence contributions are prerequisite to decipher the Xcm-cotton interactions.


In this study, we identified six tal genes in Xss-V2–18, a highly-virulent strain of Xcm from China, and assessed their role in BBC. RFLP-based Southern hybridization assays indicated that Xss-V2–18 harbors the six tal genes on a plasmid. The plasmid-encoded tal genes were isolated by cloning BamHI fragments and screening clones by colony hybridization. The tal genes were sequenced by inserting a Tn5 transposon in the DNA encoding the central repeat region (CRR) of each tal gene. Xcm TALome evolutionary relationship based on TALEs CRR revealed relatedness of Xss-V2–18 to MSCT1 and MS14003 from the United States. However, Tal2 of Xss-V2–18 differs at two repeat variable diresidues (RVDs) from Tal6 and Tal26 in MSCT1 and MS14003, respectively, inferred functional dissimilarity. The suicide vector pKMS1 was then used to construct tal deletion mutants in Xcm Xss-V2–18. The mutants were evaluated for pathogenicity in cotton based on symptomology and growth in planta. Four mutants showed attenuated virulence and all contained mutations in tal2. One tal2 mutant designated M2 was further investigated in complementation assays. When tal2 was introduced into Xcm M2 and expressed in trans, the mutant was complemented for both symptoms and growth in planta, thus indicating that tal2 functions as a virulence factor in Xcm Xss-V2–18.


Overall, the results demonstrated that Tal2 is a major pathogenicity factor in Xcm strain Xss-V2–18 that contributes significantly in BBC. This study provides a foundation for future efforts aimed at identifying susceptibility genes in cotton that are targeted by Tal2.


Cotton (Gossypium spp.) is an economically-important crop worldwide and is a significant source of fiber, feed, oil and biofuel [1]. The primary cotton production areas are located in the southern United States (USA), Central America, western Africa, and central and eastern Asia. According to the 2017/18 world ranking, China leads the world in cotton production followed by India, the USA and Pakistan [2]. Gossypium spp. contains over 50 species, including G. arboreum, G. herbaceum, G. hirsutum and G. barbadense. G. arboretum and G. herbaceum are diploid (2n = 26), whereas G. hirsutum and G. barbadense are tetraploid (4n = 52) [3, 4]. G. hirsutum is the predominant species and produces with 90% of the world’s cotton fiber production [5]. This species is impacted by a devastating bacterial disease known as bacterial blight of cotton (BBC), which is caused by Xanthomonas citri pv. malvacearum. The first detailed description of BBC was reported in the USA [6]. However, this disease currently occurs in all cotton production areas and causes significant yield losses (5–35%) either by injury to the plant or direct damage to the boll [7].

Xcm is able to infects all above-ground parts of cotton at any developmental stage starting with seedlings [8]. Typical BBC symptoms include cotyledon/seedling blight, angular leaf spots, water-soaked lesions, black arm of petioles and stems, boll rot and boll shedding [8, 9]. The main virulence factors that contribute to the pathogenicity and adaptation of bacterial pathogens include exopolysaccharides, lipopolysaccharides, adhesins, protein secretion systems, siderophores, quorum sensing, biofilms, chemotactic sensors and degradative enzymes [10,11,12,13]. Particularly, type III secreted effector (T3SE) proteins play an important role in bacterial pathogenicity [10,11,12, 14] and have been identified in Xanthomonas spp. [14,15,16,17,18,19,20]. One of the most studied groups of T3SEs are the transcriptional-activator like (tal) effector (TALE) proteins [21,22,23,24,25,26,27,28].

TALE proteins, functionally resemble eukaryotic transcription factors, are localized to the host plant nucleus where they bind to specific promoter sequences known as effector-binding elements (EBEs), thus regulating host gene expression [29,30,31]. TALEs belong to the avrBs3/pthA gene family [26], which is highly conserved among different Xanthomonas spp. TALEs contain an N-terminal T3S signal domain, a central repeat region (CRR), C-terminal nuclear localization signals (NLS), and an acid activation domain (AD) [30, 31]. CRRs contain tandem repeats of 33–35 amino acids that differ only at residues 12 and 13; these are designated repeat variable di-residues (RVDs) and determine the specificity of DNA binding [30,31,32]. TALE-mediated activation of EBEs can induce host susceptibility (S) or resistance (R) genes [29, 30]. For example, the TALEs PthXo1 and PthXo2 from X. oryzae pv. oryzae (Xoo) were shown to enhance the expression of rice genes OsSWEET11 and OsSWEET13, which are required for susceptibility to bacterial leaf blight [33, 34]. However, rice cultivars were resistant to Xoo when they contained OsSWEET11 and OsSWEET13 alleles lacking PthXo1 and PthXo2 EBEs [35,36,37]. Recently, a new rice S gene (OsERF#123) was shown to be targeted by TalB in African strains of Xoo [38]. Other examples of TALEs include AvrBs3 that targets the pepper resistance gene Bs3 and AvrXa10, AvrXa23 and AvrXa27 that interact with rice R genes Xa10, Xa23 and Xa27, respectively [29, 39,40,41]. Recently, Cai et al. [21] reported that Tal7 from Xoo binds and activates the expression of the rice gene Os09g29100, an interaction that suppresses avrXa7-Xa7-mediated resistance in rice. A number of truncated TALEs (truncTALEs) and interfering TALEs (iTALEs) have also been reported in Xoo that function as suppressors of Xa1-mediated defense in rice [42, 43].

Resistance to Xcm has been identified primarily in G. hirsutum. The genetic nature of resistance to BBC was first revealed in 1939, and efforts to breed cotton plants for resistance ensued shortly thereafter [44]. About 20 major R genes or polygene complexes (B genes) participate in resistance to BBC in cotton [7, 8]. Based on their virulence phenotype in differential cotton hosts, Xcm strains have been classified into 22 races that are named 1–22 [7]. Race 18 is the most common variant and was first isolated in 1973 [45, 46]. In some cases, the outcome of interactions between Xcm strains and differential cotton varieties is dependent on the avrBs3/pthA gene family in Xcm, which indicates that Xcm-cotton interactions follow the gene-for-gene model for host plant resistance [7, 10, 47, 48].

The number and diversity of tal effector genes varies among different species, pathovars and strains of Xanthomonas. For example, Xoo strains harbor 8–26 TALEs [49,50,51,52,53], Xoc strains contain 19–28 [49, 54, 55], Xtt strains contains 5–12 [56], Xtu strains contains 7–8 [56,57,58] and Xcm strains harbor 8–12 genes encoding tal effectors [27, 46, 59]. Some Xanthomonas spp. lack tal effector genes, such as X. citri pv. raphani strain 756C [54]. To date, at least 20 TALEs have been cloned and characterized from Xcm strains [25, 26, 28, 48]. Among these, Avrb6 was the first Xcm TALE shown to be important for virulence [25]. Cox et al. [27] demonstrated that Avrb6 induced the expression of the cotton S gene, GhSWEET10, thus enhancing bacterial virulence and adaptation to the host.

The aim of the current study is to identify a novel virulent tal-gene encoding TALE protein in a highly virulent cotton pathogen, Xcm strain Xss-V2–18 (from China).


TALEs of Xss-V2–18

Restriction fragment length polymorphism (RFLP) analysis was conducted to estimate the number and size of tal genes in Xcm Xss-V2–18. Since most tal genes retain two BamHI sites, Xcm Xss-V2–18 plasmid and genomic DNAs were digested with BamHI and analyzed by Southern blotting as described above. Six bands hybridized to the probe in BamHI-digested genomic and plasmid DNA, indicating that Xss-V2–18 contained six plasmid-encoded tal genes (Fig. 1a).

Fig. 1
figure 1

Southern blotting, and Isolation and sequencing of Xss-V2–18 tal-genes. a Southern blot analysis of BamHI-digested genomic (gDNA) and plasmid DNA (pDNA) of Xcm strain Xss-V2–18. A 2.9-kb SphI fragment of pthXo1 (from Xoo) was labeled with digoxygenin (DIG) and used as a probe to detect tal genes in Xcm Xss-V2–18. b Plasmid DNA of Xss-V2–18 was digested with BamHI, and fragments were gel-purified and ligated into BamHI-digested and CIP-treated pBluescript II SK(−). Southern blot analysis was performed by the using internal SphI fragment of pthXo1 as a probe to confirm each clone (pB-tal1 – pB-tal6). c Schematic diagram of strategy used to sequence tal genes. After cloning into pBluescript II SK(−), the EZ-Tn5™ < KAN-2 > Tnp Transposome™ Kit was used to insert Tn5 into each tal gene. Clones with Tn5 insertions in the middle of the CRR were selected by SphI digestion and sequenced using primer pairs tal-F/RP and FP/tal-R

The six tal genes were cloned in pBluescript as BamHI fragments, giving rise to pB-tal1, pB-tal2, pB-tal3, pB-tal4, pB-tal5 and pB-tal6 (Fig. 1b) and confirmed by colony hybridization and sequence analysis. To obtain the complete DNA sequence of each tal gene, we inserted the Tn5 transposon into the CRR region and used primer sets tal-F/RP and FP/tal-R to obtain the sequences (Fig. 1c). The tal gene sequences have been deposited in GenBank under the following accession numbers: MK654746 (tal1), MK654747 (tal2), MK654748 (tal3), MK654749 (tal4), MK654750 (tal5) and MK654751 (tal6). Each tal gene encodes various numbers of RVDs, which are tandemly arranged and encoded within 102-bp direct repeats. There were 27.5, 102-bp repeat units in tal1, 25.5 in tal2, 21.5 in tal3, 18.5 in tal4, 15.5 in tal5 and 13.5 in tal6 (Fig. 2a).

Fig. 2
figure 2

Alignment of TALE RVDs and TALEs Phylogeny. a Alignment of TALE RVDs from Xcm strains Xss-V2–18, MSCT1, H1005, MS14003 and AR81009 with AnnoTALE (version 1.4.1). Letters in red font indicate RVDs that differ between the two strains. The asterisk represents a missing amino acid residue b Construction of phylogenetic tree based on central repeat amino acid sequences of TALEs. A set of 53 TAL effector sequences from 6 different Xcm strains were used to construct tree with DisTAL program using default parameters. TALEs were classified into 6 major groups and 33 sub-groups showing the relationship of Xcm Xss-V2–18 to other Xcm strains published previously. Tal2 of Xss-V2–18, TAL6 of MCST and Tal26 (M26) of MS14003 fall in same group. Scale is shown below the tree

To better understand the features of Xss-V2–18 TALEs, we compared them with TALEs in Xcm strains MSCT1, H1005, N1003, MS14003 and AR81009 [27, 46, 59]. Phylogenetic tree of TALEs from Xcm strains were constructed by aligning TALE-CRR with DisTAL v1.1. All 53 TALEs (Xss-V2–18 = 6, MSCT1 = 8, H1005 = 12, N1003 = 9, MS14003 = 8 and AR81009 = 12) were classified into 6 major groups and 33 sub-groups. Tal2 of Xss-V2–18, TAL6 of MCST and Tal26 of MS14003 fall in same group (Fig. 2b).

Nearly identical RVD sequences were observed for the six TALEs in Xss-V2–18, MSCT1, H1005, MS14003 and AR81009 (Fig. 2a). Differences of two RVDs between Tal2 of Xss-V2–18 and TAL6 of MSCT1, Tal26 of MS14003 indicate that they are functionally different from each other and may target a different EBE. The predicted theoretical EBE box for Tal2, Tal6 and Tal26 of Xss-V2–18, MSCT1 and MS14003, respectively, are mentioned in Fig. S1. RVDs in Xcm strains included NI, NG, NS, HD and NN; the latter RVD was absent in Tal1, Tal2, Tal3 and Tal4.

Xss-V2–18 tal deletion mutants

To assess the role of tal genes in the virulence of Xss-V2–18, we generated tal deletion mutants by homologous recombination using the suicide vector pKMS1 [60]. Fragments a (580 bp) and b (350 bp) were amplified on the left and right sides of DNA encoding the CRR, respectively, and cloned as a fused fragment in pKMSA1 (Fig. 3a, b). Construct pKMSA1 was introduced into Xcm Xss-V2–18; after homologous recombination, 41 putative mutants were selected for PCR amplification using primers pKMSA1-5F/pKMSA1-3R (Table S1). Four putative mutants designated M1, M2, M3 and M4 contained a 930-bp PCR product, which is consistent with the size of the insert in pKMSA1 (Table 1, Fig. 3c). Southern hybridization indicated that one or more tal genes were deleted in the four mutants (Fig. 3d). M1 and M2 were lacking tal3 and tal2, respectively, M3 was missing tal2 and tal4, and M4 lacked tal2, tal4, tal5, and tal6. These results indicated that four tal loci underwent homologous exchange via pKMSA1, and copies of the plasmid pKMSA1 functioned to delete multiple tal genes simultaneously in M3 and M4.

Fig. 3
figure 3

Deletion mutagenesis of Xss-V2–18 tal genes. a Schematic diagram of suicide plasmids pKMSA1 and pKMSA2. Fragments a (580 bp) and b (350 bp) were amplified on the left and right sides of the CRR, respectively, and cloned as a fused fragment in pKMSA1. Fragments c (580 bp) and d (150 bp) were amplified on the left and right sides of the CRR, respectively, and cloned as a fused fragment in pKMSA2. Constructs pKMSA1 and pKMSA2 were introduced into Xcm strain Xss- V2–18 by electroporation, and deletion of the CRR region was conducted as described in Methods. b Confirmation of 930- and 450- bp inserts in pKMSA1 and pKMSA2, respectively, by digestion with XbaI and SmaI. c PCR analysis of 41 putative mutants with primers pKMSA1-5F and pKMSA1-3R. A 930-bp fragment was amplified in M1, M2, M3, and M4, indicating that these four mutants underwent a homologous recombination and potential deletion of the CRR region. pKMSA1 was included as a control. d Southern hybridization analysis of Xss-V2–18 and mutant strains M1-M4. Plasmid DNA of WT Xss-V2–18 and mutants were isolated and digested with BamHI. The internal SphI fragment of pthXo1 (from Xoo) was used as a hybridization probe to detect tal genes. e PCR screening for putative mutants using primers pKMSA2-5F and pKMSA2-3R. pKMSA2 was included and used as a positive control. f Southern hybridization analysis of mutant M4 (used for second round of mutagenesis), M5 and M6. Plasmid DNA of M4, M5 and M6 were isolated and digested with BamHI, and the internal SphI fragment of pthXo1 was used a hybridization probe to detect tal genes

Table 1 List of strains and plasmids used in this study

A second round of deletion mutagenesis was conducted with plasmid pKMSA2, which contains a fusion of fragments c (150 bp) and d (300 bp) on the left and right sides of the DNA encoding the CRR, respectively (Fig. 3a). Construct pKMSA2 was used to generate new deletions in the M4 mutant, and potential new mutants were analyzed by PCR with primer pairs pKMSA2-5F/pKMSA2-3R (Table S1). Two mutants designated M5 and M6 contained a 450-bp PCR product that is consistent with the size of the insert in pKMSA2 (Fig. 3e). In addition to tal2, tal4, tal5, and tal6, Southern hybridization indicated that mutant M5 contained a deletion in tal3. M6 was lacking both tal1 and tal3 (Fig. 3f); thus, M6 lacked all six tal genes and can be considered a tal-free mutant of Xss-V2–18.

Virulence assays

Xss-V2–18 and mutants M1-M6 were inoculated into cotton leaves and phenotypes were observed 3–5 days post-inoculation (Fig. 4a). Xss-V2–18, M1, and M4 produced substantial water-soaked lesions in the inoculation sites; however, water-soaking was reduced in leaves inoculated with M2, M3, and M5 (Fig. 4a). In contrast, the region inoculated with the tal-free mutant M6 showed cell death and necrosis (Fig. 4a) signifying that the loss of tal genes affect the virulence of Xss-V2–18. On the second day post-inoculation, the populations of the M2 and M6 mutants were significantly lower than Xss-V2–18, M1, M3, M4 and M5 (Fig. 4b). On days 4 and 6 post-inoculation, the growth of Xss-V2–18 was significantly higher than mutants M1-M6 with no significant difference among the mutants. These results indicated that some of the tal genes are involved in Xss-V2–18 virulence, and the absence of selected tal genes impacted growth of the pathogen in planta.

Fig. 4
figure 4

Tal2 contribution to virulence of Xss-V2–18 on cotton variety TM-1. a Phenotypes of the mutant strains relative to wild-type Xss-V2–18. Wild-type (WT) and mutant strains were inoculated to the lower surface of cotton leaves (two-week-old plants) using a needleless syringe. Infiltration with simply 10 mM MgCl2 served as a mock. Phenotypes were observed 3–5 days post-inoculation. bIn planta growth of WT Xss-V2–18 and mutants. Growth was measured at 0, 2, 4, and 6 days post-inoculation. Error bars represent means and standard deviations (means ± SD), and columns labeled with different letters represent significant differences (P < 0.05). c Western blot analysis of TALE production in Xcm M2. Plasmid pHZW-tal2 was transferred into Xcm M2 by electroporation. Production of TALE was analyzed by western blotting using an anti-FLAG primary antibody (see Methods). RNA polymerase subunit alpha (RNAP) from E. coli, was used as a loading control. d Symptoms in cotton leaves inoculated with Xss-V2–18, mutant M2, M2 containing empty vector and M2 containing tal2 in trans. Bacterial strains were inoculated to cotton leaves using a needleless syringe, and phenotypes were observed within 5–7 days post-inoculation. eIn planta growth of the WT Xss-V2–18, mutant M2 and complemented strain. Growth was measured at 0, 2, 4, and 6 days post-inoculation. Error bars represent means and standard deviations (means ± SD), and columns labeled with different letters represent significant differences (P < 0.05)

Mutant M2, which lacks tal2, exhibited reduced symptomology and bacterial growth when compared to wild-type Xss-V2–18 (Fig. 4a, b). Based on these observations, we speculated that tal2 might be involved in virulence; this was addressed by constructing pHZW-tal2 (Table 1) for complementation analysis. The pHZW-tal2 construct was introduced into Xcm M2, and the empty vector (ev, pHM1) was used as a negative control. Western blot analysis indicated that the Tal2 protein was produced in Xcm M2 (Fig. 4c). The wild-type Xss-V2–18, mutant M2, M2(ev), and M2(tal2) were inoculated into cotton leaves; phenotypes were observed at 5–7 days post-inoculation (Fig. 4d), and bacterial growth was measured at 0, 2, 4, and 6 days post-inoculation (Fig. 4e). Both water-soaking and bacterial growth in planta were restored to wild-type levels in Xcm M2 containing pHZW-tal2 (Fig. 4d, e). Based on results shown in Fig. 4, we conclude that Tal2 is major virulence factor in Xss-V2–18.


Until recently, BBC has been effectively controlled using classical R genes [63,64,65]; however, in 2011 the disease re-emerged with a vengeance [46]. A known virulence factors, transcription activator-like effectors (TALEs), in Xcm are important for BBC. In previous studies, 8–12 tal genes were reported in Xcm [26,27,28, 48, 59]. Some Xcm tal genes, notably avrB101, avrB102 and avrBln, are known to cause an hypersensitive response (HR) on cotton [28], whereas avrb6 elicits water-soaking [48]. In this study, RFLP-based Southern hybridizations indicated that the highly-virulent Xcm strain Xss-V2–18, which was originally isolated from Hainan, China, harbors six plasmid-borne tal genes (Fig. 1). In the genus Xanthomonas, the location and number of tal genes varies among species, pathovars and strains [55, 66]. For example, strains of X. oryzae pv. oryzicola (Xoc) encode over 250 chromosomally-borne tal genes [55]; however, plasmid-encoded tal genes are common in other Xanthomonas spp. Examples include the tal genes in X. citri pv. citri, X. citri pv. aurantifolii and X. axonopodis pv. manihotis, which were identified on plasmids pXAC66, pXcB and pXam46, respectively [67,68,69]. Feyter and Gabriel [28] and Showmaker et al. [59] reported the existence of plasmid-borne tal genes in Xcm strains XcmH and MSCT1, respectively. A draft genome sequence of the Xanthomonas translucens pv. cerealis strain CFBP 2541 also indicate a plasmid borne tal-gene [70].

The presence of highly repetitive sequences in tal genes complicates efforts to obtain their nucleotide sequence; therefore, we used a Tn5 insertion method as a sequencing strategy. This sequencing strategy for tal-genes was also used by others previously [21, 71]. Normally the number of repeats in tal genes varies between 1.5 and 33.5, and each repeat encodes 33–34 amino acids that vary only at positions 12 and 13 (RVDs) [30]. In Xcm Xss-V2–18, we identified 27.5, 25.5, 21.5, 18.5, 15.5 and 13.5 tandemly arranged 102-bp direct repeats (encoding 34 amino acids) in tal1, tal2, tal3, tal4, tal5 and tal6, respectively. In order to understand how Xcm TALome differ from each other within and between strains, DisTAL and AnnoTALE were used to characterized [50, 72]. Xcm encodes very diverse TAL effectors that were classified exclusively into 6 major groups and 33 sub-groups. TALE phylogenetic tree of Xcm strains showed that Tal2 of Xss-V2–18, TAL6 of MCST and Tal26 (M26) of MS14003 fall in same group. Furthermore, RVDs based analysis showed that the six TALEs in Xss-V2–18 were identical or nearly identical to plasmid-borne TALEs in Xcm MSCT1, MS14003, H1005 and AR81009 which suggests that these genes may have been horizontally transferred [67, 73, 74]. The number and location of tal genes varied in the six Xcm strains; MSCT1 possess eight (seven plasmid-borne) [59], XcmH1005 has 12 (six plasmid- and six chromosomally-encoded) [27], XcmN1003 has nine (four plasmid-encoded) [27], MS14003 has 8 (7 plasmid-encoded) [46], AR81009 has 12 (six plasmid-encode) [46] and Xss-V2–18 has six plasmid-encoded tal genes (Figs. 1, 2). The variation in number, location and RVD sequence in Xcm TALEs could be important for maintaining virulence in cotton cultivars grown in different geographical regions.

To assess the role of tal genes in Xss-V2–18, we generated deletions in Xss-V2–18 by homologous recombination with pKMS1 [60], which was previously used to generate deletion mutants in the rice pathogen, Xoc [75,76,77]. This is the first report where pKMS1 was used to generate tal deletion mutants in Xcm, and the basic strategy was to replace the CRR (encoded by 102-bp repeat units) with up-and downstream fragments flanking the tal genes. Using construct pKMSA1, we obtained four mutants; M1 and M2 lacked tal3 and tal2, M3 had deletions in tal2 and tal4, and M4 lacked tal2, tal4, tal5 and tal6. We speculate that tal5 and tal6 might be located in the same gene cluster. The second knockout was obtained using pKMSA2 where up- and downstream flanking fragments (homology arms) were located closer to the CRR. Mutant M4 was used as a parental strain for the deletions generated with pKMSA2, and we recovered two new mutants designated M5 and M6. In addition to tal2, tal4, tal5 and tal6, mutant M5 also lacks tal3, whereas M6 contains deletions in all six tal genes (Fig. 3). The symptoms induced by M2, M3, M5 and M6 were significantly reduced relative to the wild-type, thus indicating that one or more tal genes contribute to symptom development in Xss-V2–18. Mutants M2, M3, M5 and M6 all lack the tal2 gene; thus the potential contribution of tal2 to symptom development was further investigated. Expression of tal2 in trans restored symptoms and growth in planta to the M2 mutant, thus confirming that Tal2 is a virulence factor (Fig. 4). Although the TALE repertoire of Xcm Xss-V2–18, MSCT1, MS14003, H1005 and AR81009 is somewhat identical, Tal2 of Xss-V2–18 differs at two repeat variable diresidues (RVDs) from Tal6 in MSCT1 and Tal26 in MS14003, inferred functional dissimilarity.

TALEs functionally resemble eukaryotic transcription factors that target and regulate the expression of host genes by binding to their promoter sequences. TALE-triggered susceptibility has been well-studied, and the contribution of TALEs to virulence has been evaluated in many Xanthomonas spp. [21,22,23, 27, 57, 78,79,80,81]. For example, the TALEs PthXo1 and PthXo2 from Xoo were shown to enhance the expression of rice genes OsSWEET11 and OsSWEET13, which are required for susceptibility to bacterial leaf blight in rice [33, 34]. However, rice cultivars were resistant to Xoo when they contained OsSWEET11 and OsSWEET13 alleles lacking PthXo1 and PthXo2 EBEs [35,36,37]. A recent study by Peng et al. [82] reported that Tal8 from Xtu target and induce the expression of host gene Ta-NCED-5BS, encode enzyme required for rate-limiting step in ABA biosynthesis, to promote disease susceptibility. In another new study, Wu et al. [83] shown that TAL-effector Brg11 from Ralstonia solanacearum enhance the expression of 5́-truncated ADC (arginine decarboxylase) transcripts that subvert translational control and thereby inhibit competing pathogens. In Xcm, Avrb6 was the first TALE shown to be important for virulence [25]. Recently, the Xcm effector Avrb6 was shown to target and induce the expression of the cotton S gene, GhSWEET10, thus enhancing virulence and promoting disease [27]. The present study provides an important foundation for identifying potential S genes that interact with Tal2, which will ultimately help us develop better control strategies for BBC.


In this study, we identified genes encoding TALEs in the highly-virulent Xcm strain, Xss-V2–18 (from China), and assessed TALE roles in BBC. We found that Xss-V2–18 encodes six plasmid-borne tal genes. Knockout mutagenesis of Xss-V2–18 tal genes and complementation analysis demonstrated that Tal2 is required for full virulence of Xss-V2–18 on cotton. The identification of the Tal2 target in cotton will ultimately provide new avenues for developing BBC-resistant varieties.


Bacterial strains, growth conditions, and plasmids

The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were grown in Luria-Bertani (LB) medium (5 g yeast extract, 10 g NaCl, 10 g tryptone/L) or LB with agar at 37 °C. Xcm strains were grown in nutrient broth (NB) (1 g yeast extract, 3 g beef extract, 5 g polypeptone and 10 g sucrose/L) or NB with agar at 28 °C. Xcm transformants containing the first crossover event were grown on NAN (nutrient agar without sucrose) or NBN (NAN without agar) medium. For the second crossover event, transformants were plated on NAS agar (NAN with 10% sucrose) [60]. When appropriate, antibiotics were added at the following concentrations (μg/mL): ampicillin, 100; kanamycin, 20; spectinomycin, 25; and rifampicin, 50. The pH of both solid and liquid media was adjusted to 7.0–7.2.

DNA preparation

Total genomic DNA of Xss-V2–18 was isolated using the Bacterial Genomic DNA Extraction Kit (TaKaRa, China). The isolated gDNA pellet was re-suspended in double-distilled water. Bacterial plasmid DNA was isolated using the Plasmid Miniprep Kit (Omega, USA). The quality and quantity of genomic DNA and plasmid DNA were checked with NanoDrop spectrophotometer (Eppendorf). Routine plasmids isolation from E. coli was carried out by using the plasmid DNA Mini Kit (GBS Biotechnology, China).

Isolating, cloning and sequencing of Xss-V2–18 tal genes

The isolation and cloning of tal genes from Xcm strain Xcc-V2–18 followed a previously described procedure [21, 71, 79, 84] with minor modifications. Plasmid DNA and genomic DNA (50 μg) were isolated from Xcm, digested with BamHI, and separated on 1.2% agarose gels. Specific tal DNA fragments were then gel-purified and ligated into pBluescriptII SK(−) that was digested with BamHI and treated with calf intestinal phosphatase (CIP). The ligated products were introduced into competent E. coli cells by the heat shock method according to the manufacturer’s protocol (Bio-Rad, USA). The successful cloning of tal genes in pBluescript II was validated by restriction digestion, colony hybridization and sequence analysis.

The repeat units in tal genes complicate abilities to sequence the genes using conventional approaches. Thus, after cloning into pBluescript II SK(−), we used the EZ-Tn5™ < KAN-2 > Tnp Transposome™ Kit to insert Tn5 into each tal gene as recommended by the manufacturer (Epicentre, Madison, WI). Clones with Tn5 insertions in the middle of the repeat region were selected by SphI digestion and sequenced using primers pair tal-F/RP and FP/tal-R (Table S1).

TALEs phylogenetic tree construction and RVDs comparison

For TALEs phylogeny, available genome sequences of Xcm strains MSCT1, H1005, N1003, MS14003 and AR81009 were obtained from the NCBI. TALE genes were predicted and analyzed in each genome using AnnoTALE v1.4.1 [50]. DisTAL v1.1 were used to align and classify TALEs based on their central repeat region [72].

For the TALE RVDs analysis, we used AnnoTALE version 1.4.1. The TALEs are grouped into classes based on the RVDs that shows possible functional and evolutionary relationship [50, 85].

Construction of Xss-V2–18 tal deletion mutants

The tal genes in Xcm Xss-V2–18 were deleted by homologous recombination using the suicide vector pKMS1 [60]. The 5 and 3 fragments that flank the CRR repeat in tal genes are conserved [66] and were used as sites for homologous recombination. The left- and right-flanking fragments of each tal gene were PCR-amplified using genomic DNA of Xcc strain Xcc049 (Table 1) as the template, and ligated into the MCS of pKMS1 [4], resulting in constructs pKMSA1 and pKMSA2, respectively. The new constructs were verified by restriction digestion and sequence analysis (TaKaRa, China). Constructs pKMSA1 and pKMSA2 were introduced into Xcm strain Xss-V2–18 by electroporation; cells were then plated on NAN medium supplemented with kanamycin and incubated at 28 °C for 4 days. Single colonies were then cultured in NBN broth at 28 °C to OD600 ≤ 0.2 (~ 3 × 108 cells/mL), inoculated to NAS agar medium, and incubated for 2 days at 28 °C. Single colonies that grew on NAS were then transferred to NA and NA containing kanamycin. Colonies that grew on NA, but not on NAKm, were selected as potential deletion mutants. The mutants were then analyzed by Southern blot hybridization and PCR with primer pairs pKMSA1-5F/pKMSA1-3R and pKMSA2-5F/pKMSA2-3R (Table S1).

Southern hybridizations

Xcm plasmid and genomic DNA were extracted as described above. After BamHI digestion, DNA was separated on 1.2% agarose gels and then transferred onto Hybond N+ nylon membranes (Roche, Germany). The 2898-bp internal SphI fragment of pthXo1 (GenBank accession no: AY495676) from Xoo [86] was labeled with digoxigenin (DIG) and used as a hybridization probe to detect the tal genes. Probe labeling and Southern blotting were performed using the DIG Probe Synthesis Kit as recommended by the manufacturer’s instructions (Roche, Sweden).

Virulence assays

Cotton cultivar TM-1 (G. hirsutum) was used in this study. Plants were grown in a greenhouse at 23 °C with a 12-h light/dark photoperiod and ~ 80% RH. Two-week-old plants were used in virulence assays. Single colonies of Xcm were inoculated to 4 mL NB and cultured overnight at 28 °C. Bacterial cells were harvested by centrifugation (5000 rpm, 3 min); pellets were washed twice in sterile 10 mM MgCl2 and then re-suspended in 10 mM MgCl2 buffer to OD600 = 0.1 (~ 2 × 108 cells/mL). The suspensions were inoculated to the abaxial surface of leaves by infiltration with a sterile needleless syringe. Inoculation with simply 10 mM MgCl2 buffer served as a mock. Leaf phenotypes were examined 4–5 days after inoculation. Three independent plants were used, and the experiments were repeated three times with similar results. For the quantification of bacterial growth in cotton, triplicate leaf samples (1cm2 in diameter) were collected for each inoculated strain and washed with 70% ethanol and double-distilled water (ddw). Samples were macerated in 1 mL ddw and incubated for 30 min at room temperature. Serial dilutions were then plated on NB agar medium with appropriate antibiotics for colony counts. The experiment was repeated three times, and the significant differences were determined by using student’s t-test.

Expression of tal2 gene in Xcm M2

The plasmid pZWavrXa7 (supplied by Dr. Bing Yang) was used to construct the plasmid for expression of tal2 in Xss-V2–18 strain. Plasmid pZWavrXa7 contains a FLAG-tag epitope immediately downstream of the second SphI site in the C-terminus of AvrXa7. The central SphI fragment of avrXa7 was replaced with the SphI fragment of Xss-V2–18 tal2 gene to generate pZW-tal2 (Table 1). The recombinant plasmid was then fused with broad-host-range vector pHM1 at the HindIII site giving rise to pHZW-tal2. The constructs were transformed into Xcm M2 (∆tal2 strain, see below) by electroporation (2.5 kv, 4 ms).

The expression of tal2 in M2 was confirmed by western blotting with flag-labelled antisera. Briefly, the M2 strain containing pHZW-tal2 was cultured in NB to the logarithmic phase and harvested by centrifugation. The pellets were washed twice, and re-suspended in 1X PBS buffer to OD600 = 1.0 (~ 3 × 109 cells/mL). SDS loading buffer (5X) was added to the bacterial suspensions and boiled in a water bath for 10 min. Proteins were separated on 8% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes for immunoblotting using anti-FLAG (TransGene, Beijing, China) as the primary antibody. Primary antibodies were detected using goat anti-mouse IgG (H + L) (TransGen) and visualized with the EasySee Western Kit (TransGen). E. coli RNA polymerase subunit α (RNAP) was used as a loading control.

Availability of data and materials

All the dataset generated or analyzed during this study are included in this published article. The nucleotide sequences have been deposited in GenBank under the following accession numbers (MK654746-MK654751). The plasmids are available from the corresponding author on reasonable request.



Bacterial Blight of Cotton

Xcm :

Xanthomonas citri pv. malvacearum


Transcription Activator-Like Effector

tal :

transcription-activator like


Restriction Fragment Length Polymorphism


Central Repeat Region


Repeat Variable Diresidues


Effector Binding Element


Type III Secreted Effector


Nuclear Localization Signals


Activation Domain




Hypersensitive Response




  1. Sunilkumar G, Campbell LM, Puckhaber L, Stipanovic RD, Rathore KS. Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proc Natl Acad Sci U S A. 2006;103(48):18054–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. Sheth K. Top Cotton Producing Countries In The World. WorldAtlas. 2017. Accessed 10 Dec, 2019.

  3. Gotmare V, Singh P, Tule B. Wild and cultivated species of Cotton. In: Technical Bulletin; Central Institute for Cotton Research: Nagpur, India. 2000;5.

  4. Li F, Fan G, Wang K, Sun F, Yuan Y, Song G, Li Q, Ma Z, Lu C, Zou C, et al. Genome sequence of the cultivated cotton Gossypium arboreum. Nat Genet. 2014;46(6):567–72.

    CAS  PubMed  Article  Google Scholar 

  5. Holm F, Lindgren S. Impacts of Fairtrade on small-scale cotton producers-a field study in rural India. 2017. Accessed 12 Dec, 2019. In..

  6. Atkinson GF. Black" rust" of Cotton. In. vol. 27: Agricultural Experiment Station of the Agricultural and Mechanical College; 1891.

  7. Delannoy E, Lyon B, Marmey P, Jalloul A, Daniel J, Montillet J, Essenberg M, Nicole M. Resistance of cotton towards Xanthomonas campestris pv. malvacearum. Annu Rev Phytopathol. 2005;43:63–82.

    CAS  PubMed  Article  Google Scholar 

  8. Hillocks R. Bacterial blight. In: Cotton diseases 1992: 39–85.

  9. Al-Mousawi AH, Richardson P, Essenberg M, Johnson W. Ultrastructural studies of a compatible interaction between Xanthomonas campestris pv. malvacearum and cotton. Phytopathology. 1982;72(9):1222–30.

    Article  Google Scholar 

  10. Büttner D, Bonas U. Regulation and secretion of Xanthomonas virulence factors. FEMS Microbiol Rev. 2010;34(2):107–33.

    PubMed  Article  CAS  Google Scholar 

  11. Pfeilmeier S, Caly DL, Malone JG. Bacterial pathogenesis of plants: future challenges from a microbial perspective: challenges in bacterial molecular plant pathology. Mol Plant Pathol. 2016;17(8):1298–313.

    PubMed  PubMed Central  Article  Google Scholar 

  12. Melotto M, Kunkel BN. Virulence strategies of plant pathogenic bacteria. Springer: The Prokaryotes; 2013. p. 61–82.

    Google Scholar 

  13. Benali S, Mohamed B, Eddine HJ. Virulence strategies of phytopathogenic bacteria and their role in plant disease pathogenesis. Afr J Microbiol Res. 2014;8(30):2809–15.

    Article  Google Scholar 

  14. Xiao-bo X, Li-fang Z, Ma W-x, Zhi-yang L, Gong-you C. Identification of 17 HrpX-Regulated Proteins Including Two Novel Type III Effectors, XOC_3956 and XOC_1550, in Xanthomonas oryzae pv. oryzicola. PLoS One. 2014;9(3):e93205.

    Article  CAS  Google Scholar 

  15. Guo W, Cai L-L, Zou H-S, Ma W-X, Liu X-L, Zou L-F, Li Y-R, Chen X-B, Chen G-Y. The ketoglutarate transport protein KgtP is secreted through the type III secretion system and contributes to virulence in Xanthomonas oryzae pv. oryzae. Appl Environ Microbiol. 2012;AEM:07997–11.

    Google Scholar 

  16. Furutani A, Takaoka M, Sanada H, Noguchi Y, Oku T, Tsuno K, Ochiai H, Tsuge S. Identification of novel type III secretion effectors in Xanthomonas oryzae pv. oryzae. Mol Plant-Microbe Interact. 2009;22(1):96–106.

    CAS  PubMed  Article  Google Scholar 

  17. Cui Y, Zou L, Zou H, Li Y, Zakria M, Chen G. HrpE3 is a type III effector protein required for full virulence of Xanthomonas oryzae pv. oryzicola in rice. Mol Plant Pathol. 2013;14(7):678–92.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Guo W, Cui Y-p, Li Y-r, Che Y-z, Yuan L, Zou L-f, Zou H-s, Chen G-y. Identification of seven Xanthomonas oryzae pv. oryzicola genes potentially involved in pathogenesis in rice. Microbiology. 2012;158(2):505–18.

    CAS  PubMed  Article  Google Scholar 

  19. Ji Z, Wang C, Zhao K. Rice routes of countering Xanthomonas oryzae. Int J Mol Sci. 2018;19(10):3008.

    PubMed Central  Article  CAS  Google Scholar 

  20. Shah SMA, Haq F, Ma W, Xu X, Wang S, Xu Z, Zou L, Zhu B, Chen G. Tal1NXtc01 in Xanthomonas translucens pv. cerealis Contributes to Virulence in Bacterial Leaf Streak of Wheat. Front Microbiol. 2019;10:2040.

    PubMed  PubMed Central  Article  Google Scholar 

  21. Cai L, Cao Y, Xu Z, Ma W, Zakria M, Zou L, Cheng Z, Chen G. A transcription activator-like effector Tal7 of Xanthomonas oryzae pv. oryzicola activates rice gene Os09g29100 to suppress rice immunity. Sci Rep. 2017;7(1):5089.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  22. Ma W, Zou L, Ji Z, Xu X, Xu Z, Yang Y, Alfano JR, Chen G. Xanthomonas oryzae pv. oryzae TALE proteins recruit OsTFIIAγ1 to compensate for the absence of OsTFIIAγ5 in bacterial blight in rice. Mol Plant Pathol. 2018.

  23. Ji Z-Y, Zakria M, Zou L-F, Xiong L, Li Z, Ji G-H, Chen G-Y. Genetic diversity of transcriptional activator-like effector genes in Chinese isolates of Xanthomonas oryzae pv. oryzicola. Phytopathology. 2014;104(7):672–82.

    CAS  PubMed  Article  Google Scholar 

  24. Peng Z. Contribution of TAL effectors in Xanthomonas to diseases of rice and wheat. Kansas State University; 2015.

  25. Yang Y, Yuan Q, Gabriel DW. Watersoaking function (s) of XcmH1005 are redundantly encoded by members of the Xanthomonas avr/pth gene family. Mol Plant-Microbe Interact. 1996;9(2):105–13.

    CAS  Article  Google Scholar 

  26. Chakrabarty P, Duan Y, Gabriel D. Cloning and characterization of a member of the Xanthomonas avr/pth gene family that evades all commercially utilized cotton R genes in the United States. Phytopathology. 1997;87(11):1160–7.

    CAS  PubMed  Article  Google Scholar 

  27. Cox KL, Meng F, Wilkins KE, Li F, Wang P, Booher NJ, Carpenter SC, Chen L-Q, Zheng H, Gao X. TAL effector driven induction of a SWEET gene confers susceptibility to bacterial blight of cotton. Nat Commun. 2017;8:15588.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Feyter RD, Gabriel DW. At-least six avirulence genes are clustered on a 90-kilobase plasmid in Xanthomonas campestris pv. malvacearum. Mol Plant-Microbe Interact. 1991;4(5):423–32.

    Article  Google Scholar 

  29. Römer P, Hahn S, Jordan T, Strauß T, Bonas U, Lahaye T. Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene. Science. 2007;318(5850):645–8.

    PubMed  Article  CAS  Google Scholar 

  30. Boch J, Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu Rev Phytopathol. 2010;48:419–36.

    CAS  PubMed  Article  Google Scholar 

  31. Boch 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(5959):1509–12.

    CAS  PubMed  Article  Google Scholar 

  32. Deng D, Yan C, Pan X, Mahfouz M, Wang J, Zhu J-K, Shi Y, Yan N. Structural basis for sequence-specific recognition of DNA by TAL effectors. Science. 2012;335(6069):720–3.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Yang B, Sugio A, White FF. Os8N3 is a host disease-susceptibility gene for bacterial blight of rice. Proc Natl Acad Sci U S A. 2006;103(27):10503–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Zhou J, Peng Z, Long J, Sosso D, Liu B, Eom J-S, Huang S, Liu S, Vera Cruz C, Frommer WB, et al. Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. Plant J. 2015;82(4):632–43.

    CAS  PubMed  Article  Google Scholar 

  35. Yuan M, Chu Z, Li X, Xu C, Wang S. Pathogen-induced expressional loss of function is the key factor in race-specific bacterial resistance conferred by a recessive R gene xa13 in rice. Plant Cell Physiol. 2009;50(5):947–55.

    CAS  PubMed  Article  Google Scholar 

  36. Chu Z, Yuan M, Yao J, Ge X, Yuan B, Xu C, Li X, Fu B, Li Z, Bennetzen JL, et al. Promoter mutations of an essential gene for pollen development result in disease resistance in rice. Genes Dev. 2006;20(10):1250–5.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Liu Q, Yuan M, Zhou Y, Li X, Xiao J, Wang S. A paralog of the MtN3/saliva family recessively confers race-specific resistance to Xanthomonas oryzae in rice. Plant Cell Environ. 2011;34(11):1958–69.

    CAS  PubMed  Article  Google Scholar 

  38. Tran TT, Perez-Quintero AL, Wonni I, Carpenter SC, Yu Y, Wang L, Leach JE, Verdier V, Cunnac S, Bogdanove AJ, et al. Functional analysis of African Xanthomonas oryzae pv. oryzae TALomes reveals a new susceptibility gene in bacterial leaf blight of rice. PLoS Path. 2018;14(6):e1007092.

    Article  CAS  Google Scholar 

  39. Gu K, Yang B, Tian D, Wu L, Wang D, Sreekala C, Yang F, Chu Z, Wang G-L, White FF, et al. R gene expression induced by a type-III effector triggers disease resistance in rice. Nature. 2005;435(7045):1122.

    CAS  PubMed  Article  Google Scholar 

  40. Tian D, Wang J, Zeng X, Gu K, Qiu C, Yang X, Zhou Z, Goh M, Luo Y, Murata-Hori M, et al. The rice TAL effector–dependent resistance protein XA10 triggers cell death and calcium depletion in the endoplasmic reticulum. Plant Cell. 2014;tpc. 113:119255.

    Google Scholar 

  41. Wang C, Zhang X, Fan Y, Gao Y, Zhu Q, Zheng C, Qin T, Li Y, Che J, Zhang M, et al. XA23 is an executor R protein and confers broad-spectrum disease resistance in rice. Mol Plant. 2015;8(2):290–302.

    CAS  PubMed  Article  Google Scholar 

  42. Ji Z, Ji C, Liu B, Zou L, Chen G, Yang B. Interfering TAL effectors of Xanthomonas oryzae neutralize R-gene-mediated plant disease resistance. Nat Commun. 2016;7:13435.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Read AC, Rinaldi FC, Hutin M, He Y-Q, Triplett LR, Bogdanove AJ. Suppression of Xo1-mediated disease resistance in rice by a truncated, non-DNA-binding TAL effector of Xanthomonas oryzae. Front Plant Sci. 2016;7:1516.

    PubMed  PubMed Central  Article  Google Scholar 

  44. Knight R, Clouston T. The genetics of blackarm resistance. J Genet. 1939;38(1–2):133–59.

    Article  Google Scholar 

  45. Baldwin Jr C. Report of the bacterial blight committee--1975 [cotton, USA]. In: Proceedings Beltwide Cotton Production Research Conferences: 1976.

  46. Phillips AZ, Berry JC, Wilson MC, Vijayaraghavan A, Burke J, Bunn JI, Allen TW, Wheeler T, Bart RS. Genomics-enabled analysis of the emergent disease cotton bacterial blight. PLoS Genet. 2017;13(9):e1007003.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  47. Gabriel DW, Burges A, Lazo GR. Gene-for-gene interactions of five cloned avirulence genes from Xanthomonas campestris pv. malvacearum with specific resistance genes in cotton. Proc Natl Acad Sci U S A. 1986;83(17):6415–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Yang Y. Host-specific symptoms and increassed release of Xanthomonas citri and X. campestris pv. malvacearum from leaves are determined by the 102-bp tandem repeats of pthA and avrb6, respectively. Mol Plant-Microbe Interact. 1994;7:345–55.

    CAS  Article  Google Scholar 

  49. Booher NJ, Carpenter SC, Sebra RP, Wang L, Salzberg SL, Leach JE, Bogdanove AJ. Single molecule real-time sequencing of Xanthomonas oryzae genomes reveals a dynamic structure and complex TAL (transcription activator-like) effector gene relationships. Microbial genomics. 2015;1(4).

  50. Grau J, Reschke M, Erkes A, Streubel J, Morgan RD, Wilson GG, Koebnik R, Boch J. AnnoTALE: bioinformatics tools for identification, annotation, and nomenclature of TALEs from Xanthomonas genomic sequences. Sci Rep. 2016;6:21077.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. Salzberg SL, Sommer DD, Schatz MC, Phillippy AM, Rabinowicz PD, Tsuge S, Furutani A, Ochiai H, Delcher AL, Kelley D, et al. Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv. oryzae PXO99 A. BMC Genomics. 2008;9(1):204.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  52. Quibod IL, Perez-Quintero A, Booher NJ, Dossa GS, Grande G, Szurek B, Cruz CV, Bogdanove AJ, Oliva R. Effector diversification contributes to Xanthomonas oryzae pv oryzae phenotypic adaptation in a semi-isolated environment. Sci Rep. 2016;6:34137.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. Gonzalez C, Szurek B, Manceau C, Mathieu T, Séré Y, Verdier V. Molecular and pathotypic characterization of new Xanthomonas oryzae strains from West Africa. Mol Plant-Microbe Interact. 2007;20(5):534–46.

    CAS  PubMed  Article  Google Scholar 

  54. Bogdanove AJ, Koebnik R, Lu H, Furutani A, Angiuoli SV, Patil PB, Van Sluys M-A, Ryan RP, Meyer DF, Han S-W. Two new complete genome sequences offer insight into host and tissue specificity of plant pathogenic Xanthomonas spp. J Bacteriol. 2011;JB:05262–11.

    Google Scholar 

  55. Wilkins KE, Booher NJ, Wang L, Bogdanove AJ. TAL effectors and activation of predicted host targets distinguish Asian from African strains of the rice pathogen Xanthomonas oryzae pv oryzicola while strict conservation suggests universal importance of five TAL effectors. Front Plant Sci. 2015;6:536.

    PubMed  PubMed Central  Article  Google Scholar 

  56. Khojasteh M, Shah SMA, Haq F, Xu X, Taghavi SM, Osdaghi E, Chen G. Transcription Activator-Like Effectors Diversity in Iranian Strains of Xanthomonas translucens. Phytopathology. 2020;110(4):758–67.

  57. Falahi Charkhabi N, Booher NJ, Peng Z, Wang L, Rahimian H, Shams-Bakhsh M, Liu Z, Liu S, White FF, Bogdanove AJ. Complete genome sequencing and targeted mutagenesis reveal virulence contributions of Tal2 and Tal4b of Xanthomonas translucens pv. undulosa ICMP11055 in bacterial leaf streak of wheat. Front Microbiol. 2017;8:1488.

    PubMed  PubMed Central  Article  Google Scholar 

  58. Peng Z, Hu Y, Xie J, Potnis N, Akhunova A, Jones J, Liu Z, White FF, Liu S. Long read and single molecule DNA sequencing simplifies genome assembly and TAL effector gene analysis of Xanthomonas translucens. BMC Genomics. 2016;17(1):21.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  59. Showmaker KC, Arick MA, Hsu C-Y, Martin BE, Wang X, Jia J, Wubben MJ, Nichols RL, Allen TW, Peterson DG. The genome of the cotton bacterial blight pathogen Xanthomonas citri pv. malvacearum strain MSCT1. Stand Genomic Sci. 2017;12(1):42.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  60. Zou L-F, Li Y-R, Chen G-Y. A non-marker mutagenesis strategy to generate poly-hrp gene mutants in the rice pathogen Xanthomonas oryzae pv. oryzicola. Agr Sci China. 2011;10(8):1139–50.

    CAS  Article  Google Scholar 

  61. Hopkins CM, White F, Choi S, Guo A, Leach J. Identification of a family of avirulence genes from Xanthomonas oryzae pv. oryzae. Mol Plant-Microbe Interact. 1992;5(6):451–9.

    CAS  PubMed  Article  Google Scholar 

  62. Yang B, Zhu W, Johnson LB, White FF. The virulence factor AvrXa7 of Xanthomonas oryzae pv. oryzae is a type III secretion pathway-dependent nuclear-localized double-stranded DNA-binding protein. Proc Natl Acad Sci U S A. 2000;97(17):9807–12.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  63. Blank LM, Bird LS. Breeding Strains of Cotton Resistant to Bacterial Blight. Texas FARMER Collection. 1951.

  64. Green JM, Brinkerhoff L. Inheritance of three genes for bacterial blight resistance in upland cotton 1. Agron J. 1956;48(11):481–5.

    Article  Google Scholar 

  65. Simpson D, Weindling R. Bacterial blight resistance in a strain of Stoneville cotton. Agron J. 1946;38(7):630–5.

    Article  Google Scholar 

  66. Scholze H, Boch J. TAL effectors are remote controls for gene activation. Curr Opin Microbiol. 2011;14(1):47–53.

    CAS  PubMed  Article  Google Scholar 

  67. El Yacoubi B, Brunings A, Yuan Q, Shankar S, Gabriel D. In planta horizontal transfer of a major pathogenicity effector gene. Appl Environ Microbiol. 2007;73(5):1612–21.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  68. Alegria MC, Souza DP, Andrade MO, Docena C, Khater L, Ramos CH, Da Silva AC, Farah CS. Identification of new protein-protein interactions involving the products of the chromosome-and plasmid-encoded type IV secretion loci of the phytopathogen Xanthomonas axonopodis pv. Citri. J Bacteriol. 2005;187(7):2315–25.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  69. Shybut ME. Transcription Activator-Like (TAL) Effectors of the Cassava Bacterial Blight Pathogen Xanthomonas axonopodis pv. manihotis: University of California, Berkeley; 2015.

  70. Pesce C, Bolot S, Cunnac S, Portier P, Fischer-Le Saux M, Jacques MA, Gagnevin L, Arlat M, Noël LD, Carrère S et al. High-Quality Draft Genome Sequence of the Xanthomonas translucens pv. cerealis Pathotype Strain CFBP 2541. Genome Announc. 2015;3(1).

  71. Xu Z, Xu X, Gong Q, Li Z, Li Y, Wang S, Yang Y, Ma W, Liu L, Zhu B. Engineering broad-spectrum bacterial blight resistance by simultaneously disrupting variable TALE-binding elements of multiple susceptibility genes in rice. Mol Plant. 2019;12(11):1434–46.

    CAS  PubMed  Article  Google Scholar 

  72. Pérez-Quintero AL, Lamy L, Gordon J, Escalon A, Cunnac S, Szurek B, Gagnevin L. QueTAL: a suite of tools to classify and compare TAL effectors functionally and phylogenetically. Front Plant Sci. 2015;6:545.

    PubMed  PubMed Central  Article  Google Scholar 

  73. Chen NW, Serres-Giardi L, Ruh M, Briand M, Bonneau S, Darrasse A, Barbe V, Gagnevin L, Koebnik R, Jacques M-A. Horizontal gene transfer plays a major role in the pathological convergence of Xanthomonas lineages on common bean. BMC Genomics. 2018;19(1):606.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  74. Ruh M, Briand M, Bonneau S, Jacques M-A, Chen NW. Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors. BMC Genomics. 2017;18(1):670.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  75. Jiang J, Zou H, Li Y, Chen G. Expression of the hrcC, hrpE and hpa3 genes is not regulated by the hrpG and hrpX genes in a rice pathogen Xanthomonas oryzae pv. oryzicola. Acta Microbiol Sin. 2009;49(8):1018–25.

    CAS  Google Scholar 

  76. Wang Y, Zou L, Zhou D, Chen G. Key roles of hrpE gene of Xanthomonas oryzae pv. oryzicola in formation of Hrp pilus and pathogenicity in rice. Acta Phytopathol Sin. 2009;39(4):392–8.

    Google Scholar 

  77. Yanfei C, Lifang Z, Wenxiang Z, Zhiyuan J, Huasong Z, Gongyou C. Establishment of avrBs3/pthA family gene knockout system of Rice leaf spot pathogen. J Zhejiang Univ (Agric Life Sci). 2011;37(1):40–8.

    Google Scholar 

  78. Antony G, Zhou J, Huang S, Li T, Liu B, White F, Yang B. Rice xa13 recessive resistance to bacterial blight is defeated by induction of the disease susceptibility gene Os-11N3. Plant Cell. 2010;22:3864–76.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  79. Cernadas RA, Doyle EL, Niño-Liu DO, Wilkins KE, Bancroft T, Wang L, Schmidt CL, Caldo R, Yang B, White FF, et al. Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene. PLoS Path. 2014;10(2):e1003972.

    Article  CAS  Google Scholar 

  80. Cohn M, Bart RS, Shybut M, Dahlbeck D, Gomez M, Morbitzer R, Hou B-H, Frommer WB, Lahaye T, Staskawicz BJ. Xanthomonas axonopodis virulence is promoted by a transcription activator-like effector–mediated induction of a SWEET sugar transporter in cassava. Mol Plant-Microbe Interact. 2014;27(11):1186–98.

    PubMed  Article  CAS  Google Scholar 

  81. Hu Y, Zhang J, Jia H, Sosso D, Li T, Frommer WB, Yang B, White FF, Wang N, Jones JB. Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease. Proc Natl Acad Sci U S A. 2014;111(4):E521–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  82. Peng Z, Hu Y, Zhang J, Huguet-Tapia JC, Block AK, Park S, Sapkota S, Liu Z, Liu S, White FF. Xanthomonas translucens commandeers the host rate-limiting step in ABA biosynthesis for disease susceptibility. Proc Natl Acad Sci U S A. 2019;116(42):20938–46.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  83. Wu D, von Roepenack-Lahaye E, Buntru M, de Lange O, Schandry N, Pérez-Quintero AL, Weinberg Z, Lowe-Power TM, Szurek B, Michael AJ. A plant pathogen type III effector protein subverts translational regulation to boost host polyamine levels. Cell Host Microbe. 2019;26(5):638–49 e635.

    CAS  PubMed  Article  Google Scholar 

  84. Tran TT, Doucouré H, Hutin M, Niño LJ, Szurek B, Cunnac S, Koebnik R. Efficient enrichment cloning of TAL effector genes from Xanthomonas. MethodsX. 2018;5:1027–32.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  85. Erkes A, Reschke M, Boch J, Grau J. Evolution of transcription activator-like effectors in Xanthomonas oryzae. Genome Biol Evol. 2017;9(6):1599–615.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  86. Yang B, White FF. Diverse members of the AvrBs3/PthA family of type III effectors are major virulence determinants in bacterial blight disease of rice. Mol Plant-Microbe Interact. 2004;17(11):1192–200.

    CAS  PubMed  Article  Google Scholar 

Download references


We are thankful to Dr. Bing Yang, Iowa State University, for providing pHZWavrXa7.


This research work was financially supported by the National Natural Science Foundation of China (grant numbers; 31830072, 31230059). The funding bodies had no role in study design, data collection, analysis, or preparation of the manuscript.

Author information

Authors and Affiliations



GC contributed in conceiving and designing the experiments. FH and KH conducted the experiments and analyzed the data with assistance from SX, SMAS, WM, LC, XX, ZX, SW. LZ, and BZ. FH and GC wrote the paper; and all authors read, commented on, and approved the manuscript.

Corresponding author

Correspondence to Gongyou Chen.

Ethics declarations

Ethics approval and consent to participate

Not Applicable.

Consent for publication

Not Applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Additional file 1: Table S1.

Primers used in this study. Figure S1. Predicted theoretical target site logo. (A) Target site logo for Tal2 of Xss-V2-18. (B) Target site logo for Tal6 of MSCT1 and Tal26 of MS14003. Based on TALgetter (Galaxy v1.1

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Haq, F., Xie, S., Huang, K. et al. Identification of a virulence tal gene in the cotton pathogen, Xanthomonas citri pv. malvacearum strain Xss-V2–18. BMC Microbiol 20, 91 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Bacterial blight of cotton
  • Xanthomonas citri pv. malvacearum
  • Transcription-activator-like effector
  • Virulence