A Transcription Activator-Like Effector Tal7 of Xanthomonas oryzae pv. oryzicola Activates Rice Gene Os09g29100 to Suppress Rice Immunity

Abstract

Xanthomonas oryzae pv. oryzicola (Xoc) and X. oryzae pv. oryzae (Xoo) cause bacterial leaf streak (BLS) and bacterial leaf blight (BLB) in rice, respectively. Unlike Xoo, endogenous avirulence-resistance (avr-R) gene interactions have not been identified in the Xoc-rice pathosystem; however, both pathogens possess transcription activator-like effectors (TALEs) that are known to modulate R or S genes in rice. The transfer of individual tal genes from Xoc RS105 (hypervirulent) into Xoc YNB0-17 (hypovirulent) led to the identification of tal7, which suppressed avrXa7-Xa7 mediated defense in rice containing an Xa7 R gene. Mobility shift and microscale thermophoresis assays showed that Tal7 bound two EBE sites in the promoters of two rice genes, Os09g29100 and Os12g42970, which encode predicted Cyclin-D4-1 and GATA zinc finger family protein, respectively. Assays using designer TALEs and a TALE-free strain of Xoo revealed that Os09g29100 was the biologically relevant target of Tal7. Tal7 activates the expression of rice gene Os09g29100 that suppresses avrXa7-Xa7 mediated defense in Rice. TALEN editing of the Tal7-binding site in the Os09g29100 gene promoter further enhanced resistance to the pathogen Xoc RS105. The suppression of effector-trigger immunity (ETI) is a phenomenon that may contribute to the scarcity of BLS resistant cultivars.

Figure 1

Heterologous expression of avrXa7 and avrXa27 in X. oryzae pv. oryzicola (Xoc) strains YNB0-17 and RS105 and inoculation to rice cv. IR24 (susceptible) and IRBB7 (contains R gene Xa7) and 87-15 (contains R gene Xa27). (a) YNB0-17 and RS105 containing avrXa7 and avrXa27 were infiltrated into seedlings of IRBB7 (Xa7) and 87-15 (Xa27) with needleless syringes. The susceptible rice cultivar IR24 and strains containing the empty vector pHM1 were used as controls. Leaves were scored for water-soaked symptoms or HRs within the infiltrated area 2 dpi and were designated as susceptible or resistant (showing an HR). Leaves were photographed 3 dpi. (b) Strains YNB0-17 and RS105 carrying avrXa7 were inoculated to adult rice plants of IRBB7 (Xa7) and susceptible line IR24. Lesion lengths were recorded 14 dpi. The vertical columns and intersecting bars represent the mean lesion length and SD from five replicate plants. The asterisks in each horizontal data column indicate significant differences at P = 0.01 using the Student’s t test. (c) Bacterial growth in rice IRBB7 inoculated with Xoc YNB0-17 and RS105 containing avrXa7 or the empty vector control (pHM1). (d) Bacterial growth of Xoc YNB0-17 and RS105 containing avrXa7, tal7 or pHM1 in the BLS susceptible rice line IR24. Leaf discs (0.8 cm diameter) were excised from the inoculated areas, homogenized in sterile water, diluted and plated on to NA. Data points represent the mean ± SD from three replicates. All experiments were repeated three times, and similar results were obtained.

Figure 2

Tal7 suppresses avrXa7-Xa7 mediated defense. (a) Diagram showing the RVDs of Tal7 and AvrXa7 and schematic construction of Tal7∆NA. (b) Tal7 suppressesthe avrXa7-Xa7 interaction in rice. Strain YNB0-17 harboring pHM1, avrXa7, avrXa7 + tal7, and avrXa7 + tal7∆NA were infiltrated into rice seedlings of IRBB7(Xa7) with needleless syringes. The T3SS-deficient strain of RS105, R∆hrcV (Supplemental Table S1), containing avrXa7 + tal7, was used as a control. Leaves were scored for water-soaked symptoms or the HR at 3 dpi and were designated as susceptible or resistant, respectively. Leaves were photographed 3 dpi. (c) Secretion of AvrXa7-Flag and (d) Tal7-c-Myc by strain YNB0-17 harboring the constructs mentioned in panel b. Bacterial cells containing p707 or p707ΔNA (Supplemental Table S1) were induced in XOM3 medium as described in Methods. Bacterial supernatants (SN) and total extracts (TE) were analyzed by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and used for immunoblotting with anti-FLAG (c) or anti-C-Myc (d) as the primary antibody. The experiments were repeated three times, and similar results were obtained each time.

Figure 3

Xoc Tal7 targets rice genes Os09g29100 and Os12g42970. (a) The expression of candidate targets of Tal7. Xoc strains YNB0-17, and YNB0-17(tal7) were infiltrated into IR24 rice, and the expression of five rice genes (Os01g31220, Os02g14770, Os07g47790, Os09g29100, and Os12g42970) was measured 12 and 24 hpi by real time qRT-PCR. The expression levels of Actin and 18S rRNA used as internal standards. The asterisks in each horizontal data column indicate significant differences at P = 0.01 using the Student’s t test. Data are the mean ± SD of triplicate measurements from a representative experiment; and similar results were obtained in two other independent experiments. (b) Transcriptional activation of rice genes Os09g29100 and Os12g42970 by TALEs of Xoc Tal7. The TALE PthXo1 from Xoo and its target rice gene, Os8N3 (pOs8N3), were used as a positive control. Reporter fusions containing the rice promoters fused to GUS were codelivered via A. tumefaciens into N. benthamiana with (+) and without (−) constructs encoding TAL7 and PthXo1. pOs8N3, p09G29100 and p12g42970 represent the Os8N3, 09g29100 and 12g42970 promoters fused to GUS (see Methods). For quantitative assays, two leaf discs (0.9 cm diameter) were sampled 2 dpi, and GUS activity was determined using 4-methyl-umbelliferyl-β-D-glucuronide (MUG). 4-MU is 4-methyl-umbelliferone. Error bars indicate standard deviations (n = 3 samples from different plants). For qualitative assays, GUS activity in excised leaf discs was determined 3 dpi with X-Gluc (5-bromo-4-chloro-3-indolyl-b-D-glucuronide). A blue color indicatesa positive reaction. All experiments were performed twice with similar results.

Figure 4

Xoc Tal7 binds the EBEs of the Os09g29100 and Os12g42970 promoter regions. Electromobility shift assays (EMSA) were performed using 20 fM biotin-labeled DNA fragments derived from the two promoter regions as probes. Unlabeled probes were used as competitor DNA. The presence of DNA or protein is indicated by (+), and absence by (−). (a) The DNA sequences of the four probes for EMSA, 09g29100A, 09g29100B, 12g42970A and 12g42970B, are shown at the top of the panel. (b) EMSA results for the probes from Os09g29100 and Os12g42970. EBNA extract protein and biotin-EBNA coming from EMSA Kit (Thermo, USA) used as positive contral. (c) Competition of the biotinylated probes with unlabeled incubated with His-Tal7. Unlabeled probe 09g29100A and 12g42970A were incubated at increasingly higher concentrations; e.g. 5, 20, and 100 X times more than the 20 fM of biotinylated 09g29100A and 12g42970A, respectively. The experiments were repeated two or more times with similar results.

Figure 5

Os09g29100 is a major susceptibility gene in rice that can mask or suppress AvrXa7-Xa7 ETI. Strain PH, a tal-free strain of Xoo (Supplemental Table S1) was used in these experiments. (a) Expression of rice genes Os09g29100 and Os12g42970 in planta by real-time qRT-PCR. Xoo strain PH, PH/avrXa7, PH/tal7, PH/dtal3-3 and PH/dtal2-8 were infiltrated into IR24 rice leaves, and expression of Os09g29100 and Os12g42970 was evaluated 0, 12 and 24 hpi. Data are the mean ± SD of triplicate measurements from a representative experiment, and similar results were obtained in two other independent experiments. The asterisks in each column indicate significant differences at P = 0.01 by t test. (b) Role of Tal7 and two dTALEs in suppressing avrXa7-Xa7 ETI. Xoo strain PH and PH harboring avrXa7, tal7, dtal3-3 or dtal2-8 were infiltrated into rice seedlings of IRBB7 (Xa7) with needleless syringes. Images show inoculation with a single strain (five panels on left), one strain followed 3 h later with another strain (four middle panels), and two co-infiltrated strains (four panels at right). Leaves were scored for water-soaked symptoms or the HR within the infiltrated area 3 dpi and were designated as susceptible or resistant (showing an HR). Leaves were photographed 3 dpi.

Figure 6

TALEN-modified editing of EBE_tal7 in rice gene Os09g29100. (a) Colored letters indicate the nucleotide preference of Tal7 according to the TALE code. Letter height represents the preferences relative to other nucleotides for the RVD (generated with TALGetter: http://galaxy.informatik.uni-halle.de/root?tool_id=TALgetter). The RVDs of Tal7 DNA-binding domain and nucleotide sequence of the EBE_tal7 region in Os09g29100 are shown. The table shows the EBE_tal7 sequences in Nipponbare and six TALEN-edited lines (A97-1, A97-2, A97-3, A97-4, A97-5, and A97-6). The EBE_tal7 sequence is shown in red font; the dashed lines (−) indicate deletions obtained from TALEN editing. The number of T1 seeds obtained from each line is indicated. (b) Inoculation of Xoc RS105 (hypervirulent) and YNB0-17 (hypovirulent) to rice cv. Nipponbare and transgenic line A97-4 containing the TALEN-modified EBE_tal7 in Os09g29100. Strains YNB0-17 and RS105 (OD₆₀₀ = 0.5) were inoculated to adult rice plants with needleless syringes and photographed 14 dpi. (c) Disease lesion lengths on cv. Nipponbare and rice transgenic line A97-4 at 14 dpi. Column height represents mean lesion length and vertical bars show the ± SD from five replicate plants. All experiments were repeated three times, and similar results were obtained.