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. 2020 Feb 6;10(1):2036.
doi: 10.1038/s41598-020-59052-w.

TALEN-based editing of TFIIAy5 changes rice response to Xanthomonas oryzae pv. Oryzae

Affiliations

TALEN-based editing of TFIIAy5 changes rice response to Xanthomonas oryzae pv. Oryzae

Jin Han et al. Sci Rep. .

Abstract

The xa5 gene encodes a basal transcription factor (TFIIAγ) protein with wide spectrum resistance to bacterial blight caused by Xanthomonas oryzae pv. Oryzae (Xoo) in rice. It was only found in a few rice ecotypes, and the recessive characteristics limited its application in breeding. Here, we employed a TALEN-based technique to edit its dominant allelic TFIIAγ5 and obtained many mutant TFIIAγ5 genes. Most of them reduced rice susceptibility to varying degrees when the plants were challenged with the Xoo. In particular, the knocked-out TFIIAγ5 can reduce the rice susceptibility significantly, although it cannot reach the xa5-mediated resistance level, indicating TFIIAγ5 is a major component involved in disease susceptibility. In addition, the mutant encoding the protein with deletion of the 32nd amino acid or amino acid insertion between 32nd and 33rd site confers rice with the similar resistance to that of the knocked-out TFIIAγ5. Thus, the amino acids around 32nd site are also the important action sites of TFIIAγ5 besides the 39th amino acid previously reported. Moreover, the integration of xa5 into TFIIAγ5-knockout plants conferred them with a similar resistance as IRBB5, the rice variety containing the homozygous xa5 gene. Thus, TFIIAγ5 was not simply regarded as a resistant or a susceptible locus, as the substitution of amino acids might shift its functions.

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Conflict of interest statement

Dr. Xia’s work has been funded by the National Natural Science Foundation of China (No. 31071173). He has received compensation as a member of Hainan University of China. Dr. Han, Dr. Jiang and Prof. Zhai declare no potential conflict of interest.

Figures

Figure 1
Figure 1
Structure of the gene TFIIAγ5 (Xa5andxa5) displaying the discrepancy between the allelic Xa5 and xa5 that was involved in recognition by the TALEN-Xa5. The rice gene TFIIAγ5 has two natural alleles, Xa5 and xa5. The gene structure of both genes is the same and is shown above. The target region of the TALEN-Xa5 locates in the exon 1 and marked by a red strip. The underlined target sequence of TALEN-Xa5 in Xa5 and xa5 is showed in the bottom of the figure. The discrepancy of the two nucleotides between Xa5 and xa5 was also showed in red letters.
Figure 2
Figure 2
The lesions length of rice leaves in the knock-out mutants 14 days after inoculation with PXO86. (a) The T2 plants and their derivative T3 plants were inoculated by PXO86 in Beijing in August, 2015 and 2016 respectively. (b,c) The T3 plants in MH86 or D62B background were examined in Hainan in February, 2016. (d) The disease symptoms of the T3 plants were photographed 14 days after inoculation. (e) The targeted sequences of TALEN-Xa5 in transgenic plants. WT is the initial target sequence of Xa5, and the others are the mutant sequences. TP309, MH86, and D62B. TP309, MH86, and D62B are blank controls; TP, 1TP, 2TP and 3T were transgenic controls in TP309 background with pCAMBIA1300 vector; 1MH and 2MH are transgenic controls in MH86 background; 1D6 is the transgenic control in D62B background. All controls had the same target sequence as the WT. The other rice lines were knock-out mutants with their target sequences shown in e. TP, 119, 123, 278, 23 and 179 are the homozygous T2 mutant plants. 1TP, 2TP and 3TP are the random selected T3 plants derived from the TP; 119-1, 119-2 and 119-3 are the T3 plants from the 119; 123-1, 123-2, 123-3, and 123-4 are the T3 plants from the 123. The 278-1 and 278-2 are homozygous mutant T3 plants from the 278. 23-1 and 179-1 are homozygous mutant T3 plants from 23 and 179 respectively. Bars represent the average ± SD of three biological repeats. Different letters above columns indicate significant differences at P < 0.05 as determined by a one-way ANOVA followed by post hoc Tukey honest significant difference (HSD) analysis.
Figure 3
Figure 3
Resistance spectrum analyses of the Xa5 knockout mutants. The spectrum analysis was carried out in TP309 (a), D62B (b), and MH86 (c) genetic backgrounds. (d) The targeted sequences of TALEN-Xa5 in transgenic plants. The target sequence in TP309, D62B, and MH86 are same and refer as WT; 192, 294 and 23 are the homozygous T2 knockout mutants from TP309, D62B and MH86 respectively. PXO86 (P2), PXO71 (P4), PXO112 (P5), PXO145 (P7) and PXO280 (P8) are the Philippines Xoo strains; HN01 is a newly isolated Xoo strain from Hainan, China. Bars represent the average ± SD of three biological repeats. Asterisks signs indicate a statistically significant difference compared with the control plants (*P < 0.05 and **P < 0.01).
Figure 4
Figure 4
Resistance analysis of Xa5 and xa5 in the plants containing Xa21 gene. (a)The lesions length of rice leaves in the knock-out mutants 14 days after inoculation with HN01. (b) The targeted sequences of TALEN-Xa5 in transgenic plants. CX1 and CX5 are T3 knock-out mutants from CX6221B (a stable hybrid line with Xa21 gene in D62B background), CX1-xa5 and CX5-xa5 are the T0 transgenic plants with xa5 gene in CX1 and CX5 background respectively. CX6221B contains Xa5 and Xa21 gene, while IRBB5 only has the gene xa5. The HN01 was able to overcome the resistance of Xa21 gene, but not that of xa5 gene. The standard deviation (STDEV) is indicated in each column. Bars represent the average ± SD of three biological repeats. Different letters above columns indicate significant differences at P < 0.05 as determined by a one-way ANOVA followed by post hoc Tukey honest significant difference (HSD) analysis.
Figure 5
Figure 5
Lesion length in rice leaves of in-frame mutants inoculated with Xoo. (a) The homozygous T2 plants of in-frame mutants were inoculated by PXO86 in Beijing in August, 2015. TP309 and MH86 were the control plants containing the gene Xa5; TP is the T2 transgenic control plant from TP309 with pCAMBIA1300 vector. 133 is the T2 plant from TP309 with mutant Xa5 protein, in which two amino acids were replaced with six amino acids in the exon 1 (type 3 in the Table 1). The mutant Xa5 proteins in O23, O32 and 3 all have an amino acid deletion in the exon 1 (type 1 in Table 1). 217 expresses a mutant Xa5 protein with four amino acid-deletion in the exon 1 (type 5 in Table 1). 235 expresses the mutant Xa5 protein, in which five amino acids were replaced with nine amino acids in the exon 1 (type 6 in the Table 1). IRBB5 contains the homozygous resistant gene xa5, and functions as the resistant control here. (b) The homozygous T3 plants of in-frame mutants from D62B and IR24 were inoculated with PXO86 in Hainan in February 2016. D62B and IR24 are the rice varieties with homozygous Xa5 gene. 1D6 and 1IR are the T3 transgenic control plants with pCAMBIA1300 plasmid derived from D62B and IR24 respectively. 180-1 expresses a mutant Xa5 protein with three amino acid-deletion in the exon 1 (type 4 in Table 1). 260-1 expresses a mutant Xa5 protein with an amino acid insertion in the exon 1 (type 8 in Table 1). 38-1 and 38-2 are the T2 plants derived from 38 that have a nine amino acid-deletion in the exon 1 of the Xa5 protein (type 2 in Table 1). Bars represent the average ± SD of three biological repeats. Different letters above columns indicate significant differences at P < 0.05 as determined by a one-way ANOVA followed by post hoc Tukey honest significant difference (HSD) analysis.
Figure 6
Figure 6
The off-target analysis of TALEN-Xa5 in the rice TFIIAγ1 gene. (a) The possible target region of TALEN-Xa5 in the rice TFIIAγ1 gene. The target sequence in the gene Xa5 and the possible target sequence in the gene TFIIAγ1 were underlined. The restriction enzyme cutting site of SacI in the space region used to detect the mutations was marked with a blue box. The SacI can cut the PCR product into two fragments of 511 bp and 302 bp in theory. (b) The T0 generation plants of TALEN-Xa5-transformed lines in TP309 background were examined by PCR/RE. 148T0, 207T0 and 221T0 are the plants with the mutant TFIIAγ1 gene. CK1 is TP309 plant. (c) The T0 generation plants of TALEN-Xa5-transformed lines in MH86 background were examined by PCR/RE. 237 T0 is the plant with the mutant TFIIAγ1 gene. CK2 is MH86 plant. The primers of TFIIA-gamma-1F and TFIIA-gamma-1Rwere used to examine off-target activity in TFIIAγ1 were shown in Supp. Table S2.
Figure 7
Figure 7
Lesion lengths in the leaves of TFIIAγ5/Xa5 mutant plants with Xa21 gene. (a) Inoculation analysis of the T2 mutant plants from CX6221B was conducted in Beijing in August, 2015. (b) Inoculation analysis of the T3 mutant plants from CX8621 was carried out in Hainan in February, 2016. 248-1, 250-1 and 252-1 are the T3 homozygous mutants in CX8621 background from the T2 plants 248, 250 and 252, respectively. (c) The targeted sequences of TALEN-Xa5 in transgenic plants. CX6221B and CX8621 are the plants contain the homozygous resistant gene Xa21 derived from the varieties D62B and MH86 respectively. 143, 144, 146, and 147 are the T2 mutants in CX6221B background. D62B, CX6221B and CX8621 have the wild type Xa5, shown as WT in the bottom table. All the plants were inoculated with Xoo strain, HN01. Bars represent the average ± SD of three biological repeats. Asterisks signs indicate a statistically significant difference compared with the control plants (*P < 0.05 and **P < 0.01).

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References

    1. Boch J, Bonas U. Xanthomonas AvrBs3 Family-Type III Effectors: Discovery and Function. Annu. Rev. Phytopathology. 2010;48:419–436. doi: 10.1146/annurev-phyto-080508-081936. - DOI - PubMed
    1. Doyle EL, Stoddard BL, Voytas DF, Bogdanove AJ. TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA-targeting proteins. Trends Cell Biol. 2013;23:390–398. doi: 10.1016/j.tcb.2013.04.003. - DOI - PMC - PubMed
    1. Ji ZY, et al. Interfering TAL effectors of Xanthomonas oryzae neutralize R-gene-mediated plant disease resistance. Nat. Commun. 2016;7:13435. doi: 10.1038/ncomms13435. - DOI - PMC - PubMed
    1. Read AC, et al. 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. doi: 10.3389/fpls.2016.01516. - DOI - PMC - PubMed
    1. Tian DS, et al. The Rice TAL Effector-Dependent Resistance Protein XA10 Triggers Cell Death and Calcium Depletion in the Endoplasmic Reticulum. Plant. Cell. 2014;26:497–515. doi: 10.1105/tpc.113.119255. - DOI - PMC - PubMed

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