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. 2017 May 24:8:15588.
doi: 10.1038/ncomms15588.

TAL effector driven induction of a SWEET gene confers susceptibility to bacterial blight of cotton

Kevin L Cox  1   2 , Fanhong Meng  1   2 , Katherine E Wilkins  3 , Fangjun Li  1   2 , Ping Wang  1   2 , Nicholas J Booher  3 , Sara C D Carpenter  3 , Li-Qing Chen  4 , Hui Zheng  3 , Xiquan Gao  2   5   6 , Yi Zheng  7 , Zhangjun Fei  7 , John Z Yu  8 , Thomas Isakeit  1 , Terry Wheeler  1   9 , Wolf B Frommer  10 , Ping He  2   5 , Adam J Bogdanove  3 , Libo Shan  1   2
Affiliations

TAL effector driven induction of a SWEET gene confers susceptibility to bacterial blight of cotton

Kevin L Cox et al. Nat Commun. .

Abstract

Transcription activator-like (TAL) effectors from Xanthomonas citri subsp. malvacearum (Xcm) are essential for bacterial blight of cotton (BBC). Here, by combining transcriptome profiling with TAL effector-binding element (EBE) prediction, we show that GhSWEET10, encoding a functional sucrose transporter, is induced by Avrb6, a TAL effector determining Xcm pathogenicity. Activation of GhSWEET10 by designer TAL effectors (dTALEs) restores virulence of Xcm avrb6 deletion strains, whereas silencing of GhSWEET10 compromises cotton susceptibility to infections. A BBC-resistant line carrying an unknown recessive b6 gene bears the same EBE as the susceptible line, but Avrb6-mediated induction of GhSWEET10 is reduced, suggesting a unique mechanism underlying b6-mediated resistance. We show via an extensive survey of GhSWEET transcriptional responsiveness to different Xcm field isolates that additional GhSWEETs may also be involved in BBC. These findings advance our understanding of the disease and resistance in cotton and may facilitate the development cotton with improved resistance to BBC.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Comparison of whole genomes and tal genes of XcmH1005 and XcmN1003.
(a) Alignment of the XcmH1005 and XcmN1003 genomes, generated using progressiveMAUVE with default parameters. Each genome comprises a single circular chromosome and a single circular plasmid, shown linearized. Coloured, rounded rectangles represent locally collinear blocks (LCB), regions of homology without rearrangement across the aligned sequences, connected by matching coloured diagonal lines. The orientations of the LCB, forward or reverse, are indicated by their position above or below the line, respectively. The height of a column within a block reflects the average similarity relative to the other aligned sequence(s) there (see http://darlinglab.org/mauve/user-guide/viewer.html for details). Inset (bottom right) shows the alignment of the plasmids only, at a larger scale. Horizontal axes show sequence coordinates (bp). MAUVE backbone files giving the exact coordinates of all LCB are provided as Supplementary Data 1 (all molecules aligned) and Supplementary Table 1 (plasmids only). (b) The tal genes of XcmH1005 and XcmN1003. The genes are represented as block arrows at their relative positions in the chromosome or plasmid (horizontal lines). The arrows are magnified relative to the rest of the genome, but intergenic regions and arrow sizes relative to each other are to scale. Dashed lines connecting two arrows indicate identical encoded RVD sequences. Gene names follow the scheme of ref. , except for the indicated avr and pth genes, named previously. For label clarity, hypens replace ‘avr'. An apostrophe following the gene name (white arrows) indicates that the coding sequence is terminated early due to a frameshift mutation or other coding sequence disruption. Details, including RVD sequences and AnnoTALE designations, are given in Supplementary Table 2.
Figure 2
Figure 2. Transcriptome profiling coupled with EBE prediction reveals candidate target genes of Avrb6.
(a) Schematic diagram of experimental design to identify Avrb6 target genes in cotton. (b) Avrb6 protein expression in cotton protoplasts. Cotton protoplasts isolated from Ac44E were transfected with avrb6-HA or an empty vector as a control (Ctrl). Samples were collected 12 h after transfection and subjected to immunoblotting with α-HA antibody (top panel). Ponceau S. staining (Ponc.) of total protein served as the protein loading control; RuBisCo (RBC) is shown (bottom panel). (c) Avrb6 protein expression in cotton and N. benthamiana. Cotyledons from 2-week-old Ac44E cotton and leaves of 4-week-old N. benthamiana were infiltrated with Agrobacterium carrying 35S::avrb6-HA or an empty vector control (Ctrl). Inoculated tissues were collected at 48 hpi and subjected to immunoblotting. B and C were repeated three times with similar results. (d) Venn diagram of Avrb6-induced genes and genes with Avrb6 EBEs in G. raimondii genome. (e) The predicted EBE of Avrb6 RVDs. Coloured boxes on the top panel display the RVD of each repeat of Avrb6. Bottom panel indicates the relative frequencies of RVD associations with the four nucleotides for each repeat.
Figure 3
Figure 3. Avrb6 directly induces cotton gene transcription in the absence of protein synthesis.
(a) RT–PCR analysis of Avrb6 upregulated genes in cotton protoplasts. Cotton protoplasts of Ac44E were transfected with avrb6-HA or a vector control (Ctrl) and incubated for 12 h before RNA isolation. GhACTIN was used as an internal control. (b) Avrb6 contributes to water-soaking development in cotton. Cotyledons from 2-week-old Ac44E cotton were syringe-inoculated with different Xcm strains at OD600=0.1 and photographed 4 days after infiltration. Inoculation areas are indicated by dotted lines. (c) RT–PCR analysis of Avrb6 upregulated genes in cotton upon Xcm infection. Cotyledons from 2-week-old Ac44E cotton were syringe-inoculated with different Xcm strains. Tissues were collected at 12 and 24 hpi. (d) RT–PCR analysis of Avrb6 upregulated genes in the presence of CHX. Cotyledons from 2-week-old cotton were syringe-inoculated with different Xcm strains in 50 μM CHX. Tissues were collected at 24 hpi for RT–PCR. (e) Schematic diagram of the effector and reporter constructs. The reporter construct contains an expression cassette with a LUC reporter gene under the control of a candidate gene promoter. The effector construct contains either Avrb6 or PthN with an HA-epitope tag under the control of the CaMV 35S promoter. (f) Transcriptional activity of Avrb6 in cotton protoplasts. Protoplasts were co-transfected with a reporter construct and avrb6, pthN or a vector control (Ctrl), and were collected 12 h after transfection. UBQ10-GUS was included in the transfections as an internal control. The luciferase activity was normalized with GUS activity. The data are shown as mean±s.d. (n=3) from three independent repeats. Asterisks indicate significant difference using two-tailed t-test (P<0.05). (g) Schematic diagram of the transactivation assay of wild-type and mutated EBE (mEBE) in pGhSWEET10D in response to Avrb6. The nucleotide sequence containing the putative EBE is shown and the two nucleotides that were mutated are highlighted in blue. (h) Transcriptional activity of GhSWEET10D with wild type and mEBE in response to Avrb6. Cotton protoplasts were co-transfected with pGhSWEET10D::LUC carrying wild type or mEBE and Avrb6 or a vector control (Ctrl). The data are shown as mean±s.d. (n=3) from three independent repeats. The above experiments were repeated three times with similar results.
Figure 4
Figure 4. The dTALE-activating GhSWEET10 induces water-soaking in cotton.
(a) RVDs of dTALEs and the corresponding targeted EBE sequences in the G. hirsutum genome. (b) Xcm HM2.2S expressing a dTALE uniquely targeting GhSWEET10D induces water-soaking in cotton. Cotyledons from 2-week-old Ac44E plants were syringe-inoculated with different Xcm strains at OD600=0.1 and photographed at 4 dpi. The table displays the presence (+) or absence (−) of water-soaking. (c) RT–PCR analysis of GhSWEET10, GhKBS1 and GhMDR1 targeted by their respective dTALEs. Two-week-old cotyledons of Ac44E were syringe-inoculated with Xcm strains HM2.2S (OD600=0.1) containing different dTALEs. Inoculated tissues were collected at 24 hpi for RT–PCR analysis. (d) GaSWEET10 is induced by Avrb6 in G. arboreum protoplasts. Protoplasts isolated from G. arboreum were transfected with Avrb6 or an empty vector control (Ctrl) and samples were collected 12 h after transfection for RT–PCR analysis. (e) Transactivation assay of GaSWEET10 and GhSWEET10D promoters in response to Avrb6 in cotton protoplasts. Alignment of Avrb6 EBEs reveals a polymorphism between GhSWEET10D and GaSWEET10 promoters, which were highlighted in blue. Protoplasts isolated from commercial cotton variety FM706V were co-transfected with pGaSWEET10::LUC or pGhSWEET10D::LUC and Avrb6 or a vector control (Ctrl). The data are shown as mean±s.d. (n=3) from three independent repeats. (f) The dTALE matching the GaSWEET10 promoter causes water-soaking on G. arboreum. The EBE sequences of two dTALEs targeted to different regions of the GaSWEET10 promoter are shown. dTALE2 with an EBE sequence perfectly matching the GaSWEET10 promoter but not dTALE1 with a partially matching EBE sequence restores Xcm-mediated water-soaking on cotton. Cotyledons from 2-week-old G. arboreum were syringe-inoculated with different Xcm strains at OD600=0.1 and photographed at 4 dpi. The above experiments were repeated three times with similar results.
Figure 5
Figure 5. Silencing of GhSWEET10 in cotton reduces water-soaking caused by Xcm infection.
(a) Xcm-mediated water-soaking lesion development on cotton plants upon VIGS. Cotyledons from 2-week-old Ac44E plants were syringe-infiltrated with Agrobacterium carrying a VIGS construct to silence GhSWEET10 (VIGS-GhSWEET10) or a GFP vector control (VIGS-Ctrl). Three weeks later, plants were vacuum infiltrated with XcmH1005 at OD600=0.01. Images were taken 2 weeks after inoculation. (b) Quantitative analysis of Xcm-mediated water-soaking lesions on cotton plants upon VIGS. Lesions were counted on the second true leaf of each VIGS plant (at least 10 plants were inoculated for each construct in each repeat). The data are shown as mean±s.e. (n=10). The asterisk indicates a significant difference with a Student's t-test (P<0.05) when compared with the control. (c) RT–PCR analysis of GhSWEET10 expression in cotton plants upon VIGS. VIGS assays were done similarly as in a and 3 weeks later, the second true leaf was collected for RT–PCR analysis before Xcm inoculation. GhACTIN was used as an internal control. The above experiments were repeated three times with similar results.
Figure 6
Figure 6. GhSWEET10D encodes a functional clade III sucrose transporter.
(a) Detection of GhSWEET10D transporter activity in HEK293T cells using the sucrose sensor FLIPsuc90μδ3A. Sucrose transporter activity was assayed by co-expressing GhSWEET10D with the cytosolic FRET sucrose sensor FLIPsuc90μδ3A in HEK293T cells. Blue circles correspond to cells expressing the sensor alone; red circles and green circles correspond to cells co-expressing the sensor with AtSWEET11 or GhSWEET10D, respectively. The cyan block indicates duration of perfusion with 25 mM sucrose. Accumulation of sucrose is reported as a negative ratio change (mean−s.e.m.; n>10). Experiments were repeated four times with similar results. (b) Phylogenetic analysis of GhSWEET proteins from G. hirsutum. AtSWEET proteins from Arabidopsis thaliana were included to represent different clades of SWEET superfamily. The phylogenetic tree was made using the neighbour-joining method in MEGA version 6.06. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. GhSWEET10D, which was used as the query, is highlighted with a brown box. (c) Phylogenetic analysis of GhSWEET10, GhSWEET14a, GhSWEET14b and the OsSWEET proteins from Oryza sativa. The phylogenetic tree was made as described in b. The clades of the SWEET family were colour coded as follows: clade I=pink, clade II=blue, clade III=black and clade IV=orange.
Figure 7
Figure 7. Induction of GhSWEET genes in different cotton–Xcm interactions.
(a) GhSWEET10 induction by Avrb6 in protoplasts of the resistant line Acb6 and the susceptible line Ac44E. Graph displays Avrb6-induced GhSWEET10 reads per kilobase of transcript per million mapped reads and fold change from RNA-Seq analysis with Ac44E (compatible) and Acb6 (incompatible) cotton lines. (b) qRT–PCR analysis of GhSWEET10 in Ac44E and Acb6 plants upon Xcm infections. Two-week-old cotyledons were syringe-inoculated with different Xcm strains at OD600=0.1 and tissues were collected at 24 hpi. GhUBQ1 was used as an internal control. The data are shown as mean±s.d. (n=3) from three independent repeats. (c) Transactivation assay of pGhSWEET10D::LUC from Acb6 and Ac44E in response to Avrb6 in cotton protoplasts. The promoter of GhSWEET10D was amplified from Acb6 and Ac44E, respectively, and fused with luciferase reporter. Protoplasts of cotton variety FM706V were co-transfected with the reporter construct and Avrb6 or an empty vector control (Ctrl). The data are shown as mean±s.d. (n=3) from three independent repeats. An asterisk indicates the significant difference using two-tailed t-test (P<0.05) between two reporter constructs after Avrb6 induction. (d) Induction of clade III GhSWEET genes by different Xcm isolates. Two-week-old cotyledons were syringe-inoculated with different Xcm isolates (Xcm1-Xcm9) collected from different locations in Texas, USA at OD600=0.1. Tissues were collected at 24 hpi. GhUBQ1 was used as an internal control for qRT–PCR analysis. The data are shown as mean±s.d. (n=3) from three independent repeats. An asterisk indicates significant difference using two-tailed t-test (P<0.01) compared to the water control. The above experiments were repeated three times with comparable results.
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