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).
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 transposome 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. 2B).
To better understand the features of Xss-V2-18 TALEs, we compared them with TALEs in Xcm strains MSCT1, H1005, N1003, MS14003 and AR81009 [28, 46, 56]. 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=7, 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. 2A).
Nearly identical RVD sequences were observed for the six TALEs in Xss-V2-18, MSCT1, H1005, MS14003 and AR81009 (Fig. 2B). 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 Figure 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. 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. 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 (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.
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. Two mutants designated M5 and M6 contained a 450-bp PCR product that is consistent with the size of the insert in pKMSA2. 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.
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). 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.
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. 4D). 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. 4C), 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. 4C, E). Based on results shown in Fig. 5, we conclude that Tal2 is major virulence factor in Xss-V2-18.