Tal6b/AvrXa27A, a hidden TALE targeting the susceptibility gene OsSWEET11a and the resistance gene Xa27 in rice

Abstract

Xanthomonas oryzae pv. oryzae (Xoo) secretes transcription activator-like effectors (TALEs) to activate rice susceptibility (S) genes, causing bacterial blight (BB), as well as resistance (R) genes, leading to defense against BB. This activation follows a gene-for-gene paradigm that results in an arms race between the TALE of the pathogen and effector-binding elements (EBEs) in the promoters of host genes. In this study, we characterized a novel TALE, designated Tal6b/AvrXa27A, that activates the rice S gene OsSWEET11a and the rice R gene Xa27. Tal6b/AvrXa27A is a member of the AvrXa27/TalAO class and contains 16 repeat variable diresidues (RVDs); one RVD is altered and one is deleted in Tal6b/AvrXa27A compared with AvrXa27, a known avirulence (avr) effector of Xa27. Tal6b/AvrXa27A can transcriptionally activate the expression of Xa27 and OsSWEET11a via EBEs in their corresponding promoters, leading to effector-triggered immunity and susceptibility, respectively. The 16 RVDs in Tal6b/AvrXa27A have no obvious similarity to the 24 RVDs in the effector PthXo1, but EBE_Tal6b and EBE_PthXo1 are overlapped in the OsSWEET11a promoter. Tal6b/AvrXa27A is prevalent among Asian Xoo isolates, but PthXo1 has only been reported in the Philippine strain PXO99^A. Genome editing of EBE_Tal6b in the OsSWEET11a promoter further confirmed the requirement for OsSWEET11a expression in Tal6b/AvrXa27A-dependent susceptibility to Xoo. Moreover, Tal6b/AvrXa27A resulted in higher transcription of Xa27 than of OsSWEET11a, which led to a strong, rapid resistance response that blocked disease development. These findings suggest that Tal6b/AvrXa27A has a dual function: triggering resistance by activating Xa27 gene expression as an avirulence factor and inducing transcription of the S gene OsSWEET11a, resulting in virulence. Intriguingly, Tal6b/AvrXa27A, but not AvrXa27, can bind to the promoter of OsSWEET11a. The underlying recognition mechanism for this binding remains unclear but appears to deviate from the currently accepted TALE code.

Keywords: AvrXa27; OsSWEET11a; TALE; Xa27; Xanthomonas oryzae pv. oryzae; tal6b.

Figure 1

Xoo strains LN18 and LN4 do not cause disease on Xa27-containing rice. (A) Disease symptoms on rice cultivars IR24 and 78-15 after inoculation with Xoo strains PXO99^A, PH, LN18, and LN4. The images were taken 14 days post-inoculation (dpi). (B) Boxplots of mean disease lesion lengths (cm) on cvs. IR24 and 78-15. Lesions were measured at 14 dpi; dots denote individual observations from at least 5 inoculated leaves, and whiskers display the first and third quartiles, split by the median. Values with the same lowercase letters do not differ significantly at P < 0.05 according to ANOVA. (C) Genomic digest of tal genes in Xoo strains PXO99^A, LN18, and LN4 as determined by Southern blot analysis. Genomic DNA was digested with BamHI, blotted onto nylon membranes, and hybridized with the SphI fragment from avrXa27.

Figure 2

Tal6b, similar to AvrXa27, triggers Xa27 resistance. (A) Repeat variable diresidues (RVDs) in AvrXa27 and Tal6b. Single letters represent amino acids at the 12th and 13th positions of individual repeats. The asterisk indicates a predicted missing 13th residue, and RVDs in red font differ between Tal6b and AvrXa27. (B) Reaction of IR24 and 78-15 rice leaves to Xoo strains PH/ev, PH/avrXa27, and PH/tal6b. Bacterial suspensions were infiltrated into rice leaves using needleless syringes, and leaves were photographed at 3 dpi. (C) Expression of Xa27 in 78-15 rice leaves inoculated with Xoo strains. The expression of Xa27 was measured in leaves infiltrated with Xoo PH/ev (control), PH/avrXa27, and PH/tal6b. RNA was extracted from leaves at 24 hpi and used for qRT–PCR with Xa27-specific primers. Actin expression was used as an internal control. Columns labeled with two asterisks are significantly different at P < 0.01 (Student’s t-test).

Figure 3

Tal6b/AvrXa27A is a novel major virulence factor that targets the S gene OsSWEET11a. (A) Disease symptoms on rice cultivars Nipponbare and Kitaake after inoculation with Xoo strains PH/ev, PH/pthXo1, PH/avrXa27, PH/tal6b, PH/tal17, PH/tal6c, PH/tal6b-2, and PH/tal6b-6. Photos were taken at 14 dpi. (B) Boxplots of mean disease lesion lengths (cm) on Nipponbare and Kitaake. Lesions were measured at 14 dpi; dots denote individual observations from at least 5 inoculated leaves, and whiskers display the first and third quartiles, split by the median. (C) Individual RVDs of Tal6b and its derivatives Tal6b-2 and Tal6b-6. Single letters denote amino acids at the 12th and 13th positions of individual repeats. An asterisk indicates a predicted missing 13th residue. (D) Western blot analysis of TALE proteins in Xoo PH/ev, PH/tal6b, PH/tal6b-2, and PH/tal6b-6. (E) Expression of OsSWEET genes in Kitaake rice leaves inoculated with Xoo strains. The expression levels of OsSWEET11a, OsSWEET13, and OsSWEET14 were measured in rice leaves infiltrated with Xoo PH/ev and PH/tal6b. RNA was extracted from leaves 24 hpi and used for qRT–PCR with OsSWEET11a-, OsSWEET13-, and OsSWEET14-specific primers. The expression level of Actin was used as an internal control. (F) Expression of OsSWEET11a in Kitaake rice leaves inoculated with Xoo PH strains containing the empty vector (PH/ev) and AvrXa27/TalAO-class TALEs (pthXo1, tal6b/avrXa27A, tal17/avrXa27B, tal6c/avrXa27C, and avrXa27). (G) Individual RVDs in the CCR region of PthXo1 and Tal6b/AvrXa27A recognize individual nucleotides in the predicted EBE regions of the OsSWEET11a promoter. Single letters denote amino acids at the 12th and 13th positions of individual repeats, and the asterisk denotes a missing amino acid at the 13th position of a particular repeat. The scores show matches between DNA sequences in the OsSWEET11a EBE and amino acid residues in the RVDs of Tal6b/AvrXa27A and PthXo1 (positive control); scores were predicted with the TALE-NT program. Lower scores indicate higher binding affinity between the RVDs and the target sequence. EBE_PthXo1 is shown in red font, and EBE_Tal6b is underlined. (+) refers to the forward strand, and (−) refers to the reverse strand. Values with the same lowercase letters do not differ significantly at P < 0.05 according to ANOVA.

Figure 4

Tal6b/AvrXa27A binds to the EBEs of Xa27 and OsSWEET11a. (A) Nucleotide sequence of the Xa27 promoter fragment (probe); the underscored nucleotides show the EBE recognized by Tal6b/AvrXa27A. (B) His-tagged Tal6b/AvrXa27A fusion protein binds to the Xa27p probe derived from the Xa27 promoter. Positions of the bound and free probe are indicated on the left. (C) Nucleotide sequence of the OsSWEET11a promoter fragment (probe); EBE_PthXo1 is underscored. (D) His-tagged PthXo1 binds to the Xa13p probe of the OsSWEET11a promoter in gel-shift assays. Positions of the bound and free probe are indicated at the left. (E) Alignment of Xa13p, Xa13p-10, Xa13p-11, Xa13p-12, and Xa13p-13 sequences. EBE_Tal6b is underlined. Mutations in the OsSWEET11a promoter sequence are indicated in red font. (F) Binding of His-tagged Tal6b/AvrXa27A to EBE_Tal6b in the OsSWEET11a promoter.

Figure 5

OsSWEET11a promoter mutations confer resistance to Xoo. (A) DNA sequence alignment of OsSWEET11a EBE_Tal6b alleles in wild-type Kitaake rice and derived mutants. (B) Disease symptoms on representative leaves of rice lines Kitaake, MS1K-16, and MS1K-19 after inoculation with Xoo strains PH/ev, PH/pthXo1, PH/avrXa7, and PH/tal6b. (C) Boxplots of mean lesion lengths (cm) on Kitaake, MS1K-16, and MS1K-19. (D) Disease symptoms on representative leaves of Kitaake and MS1K-14 rice after inoculation with Xoo strains PH/ev, PH/pthXo1, PH/avrXa7, PH/tal6b, PH/tal6b-2, and PH/tal6b-6. (E) Boxplots of mean lesion lengths (cm) on Kitaake and MS1K-14 rice. Lesions were measured at 14 dpi. Values with identical lowercase letters do not differ significantly at P < 0.05 according to ANOVA.

Figure 6

A working model of the role of Tal6b/AvrXa27A in the interaction between Xoo and rice. pthXo1-containing Xoo strains employ the major TALE PthXo1 to specifically target the S gene OsSWEET11a, leading to ETS in rice. avrXa27-containing Xoo strains induce Xa27-mediated resistance via AvrXa27 in rice cultivars that harbor Xa27. Conversely, tal6b/avrXa27A-containing Xoo strains secrete Tal6b/AvrXa27A, which targets OsSWEET11a and Xa27. This results in Xa27-mediated ETI that suppresses OsSWEET11a-mediated ETS.