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.
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.