Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan 31;114(5):E897-E903.
doi: 10.1073/pnas.1620407114. Epub 2017 Jan 18.

TALE-induced bHLH transcription factors that activate a pectate lyase contribute to water soaking in bacterial spot of tomato

Allison R Schwartz  1 , Robert Morbitzer  2 , Thomas Lahaye  2 , Brian J Staskawicz  3
Affiliations

TALE-induced bHLH transcription factors that activate a pectate lyase contribute to water soaking in bacterial spot of tomato

Allison R Schwartz et al. Proc Natl Acad Sci U S A. .

Abstract

AvrHah1 [avirulence (avr) gene homologous to avrBs3 and hax2, no. 1] is a transcription activator-like (TAL) effector (TALE) in Xanthomonas gardneri that induces water-soaked disease lesions on fruits and leaves during bacterial spot of tomato. We observe that water from outside the leaf is drawn into the apoplast in X. gardneri-infected, but not X. gardneriΔavrHah1 (XgΔavrHah1)-infected, plants, conferring a dark, water-soaked appearance. The pull of water can facilitate entry of additional bacterial cells into the apoplast. Comparing the transcriptomes of tomato infected with X. gardneri vs. XgΔavrHah1 revealed the differential up-regulation of two basic helix-loop-helix (bHLH) transcription factors with predicted effector binding elements (EBEs) for AvrHah1. We mined our RNA-sequencing data for differentially up-regulated genes that could be direct targets of the bHLH transcription factors and therefore indirect targets of AvrHah1. We show that two pectin modification genes, a pectate lyase and pectinesterase, are targets of both bHLH transcription factors. Designer TALEs (dTALEs) for the bHLH transcription factors and the pectate lyase, but not for the pectinesterase, complement water soaking when delivered by XgΔavrHah1 By perturbing transcriptional networks and/or modifying the plant cell wall, AvrHah1 may promote water uptake to enhance tissue damage and eventual bacterial egression from the apoplast to the leaf surface. Understanding how disease symptoms develop may be a useful tool for improving the tolerance of crops from damaging disease lesions.

Keywords: TAL effector; Xanthomonas bacterial spot; water soaking.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. S1.
Fig. S1.
XgΔavrHah1 has an in-frame deletion of the DBD. (A) Schematic of the in-frame deletion that removes the DBD from avrHah1 to create avrHah1ΔDBD. NLS, nuclear localization signal. (B) Southern blot analysis of BamHI-digested genomic DNA from X. gardneri strains listed probed for the DBD (Top) or the T3SS (Bottom). The size of BamHI-digested avrHah1 is 2,964 bp, and the size of BamHI-digested DBD deletion of avrHah1 is 1,491 bp. (C) Cell death (or HR) occurs in response to activation of the Bs3 resistance gene in pepper 30R and appears here as a dark, opaque area. XgΔavrHah1 does not activate Bs3 and is pathogenic on pepper 30R. Complementation of XgΔavrHah1 with avrHah1 or avrBs3 restores activation of Bs3 resistance and HR in pepper 30R.
Fig. 1.
Fig. 1.
AvrHah1 promotes the intake of water into the apoplast of X. gardneri (Xg)-infected plants. N. benthamiana was syringe-infiltrated with X. gardneri (1), XgΔhrcV (2), XgΔavrHah1 + avrHah1 (3), and XgΔavrHah1 (4) at OD600 = 0.1 [depicted in the diagram as the bottom left area of the leaf, top left area of the leaf (2), bottom right area of the leaf (3), and top right area of the leaf (4), respectively]. At 48 hpi, a 30-μL drop of water was pipetted on top of a small epidermal wound in the infiltrated areas. Pictures were taken at the times indicated in each frame until 2 min and 20 s after application of the first water drop.
Fig. 2.
Fig. 2.
AvrHah1 induces water soaking in tomato, whereas AvrBs3 activates a hypersensitive response. Tomato Heinz 1706 leaves were syringe-infiltrated with the X. gardneri (Xg) strains indicated (OD600 = 0.1). At 48 hpi, leaves were submerged in water for 20 min, blotted with a Kimwipe, and photographed.
Fig. 3.
Fig. 3.
AvrHah1 enhances water soaked lesions in Xe85-10. Tomato leaves were infiltrated at 104 CFU/mL with Xe85-10 alone (−) or Xe85-10 complemented as indicated and observed at 6 dpi. Plants were grown at ambient humidity and were not submerged in water before observation.
Fig. S2.
Fig. S2.
XgΔavrHah1 does not experience an in planta growth defect in tomato. Tomato leaves were infiltrated with X. gardneri strains at 104 CFU/mL. At 6 dpi, six leaf discs per strain of X. gardneri were sampled for in planta bacterial growth.
Fig. 4.
Fig. 4.
Water soaking can introduce bacterial cells into the apoplast. N. benthamiana was syringe-infiltrated with either X. gardneri (Xg) or XgΔhrcV at OD600 = 0.1. Water soaking was induced at 48 hpi with a 30-μL drop of Tet-resistant X. gardneri (X. gardneri TetR, 105 CFU/mL in 1 0 mM MgCl2) on a wound (arrow). Leaf discs were collected away from the wound site after 5 min (dashed circle), ground in 10 mM MgCl2, and dilutions were plated on either Rif or Rif + Tet to select for the growth of all X. gardneri or the X. gardneri TetR from the water-soaking inoculum, respectively.
Fig. 5.
Fig. 5.
AvrHah1 and AvrBs3 are differentially recognized by tomato Bs4. Tomato leaves were infiltrated with the Xe85-10 strains indicated at OD600 = 0.1 and observed 48 hpi. Cell death is visible in response to delivery of avrBs3, whereas the beginnings of water soaking are apparent in response to avrHah1. Leaves were left at ambient humidity and were not submerged in water before observation.
Fig. S3.
Fig. S3.
AvrHah1 has unique and overlapping targets with AvrBs3. AvrHah1 (Top) can activate expression from the Bs3 promoter (proBs3) using an overlapping EBE with that of AvrBs3 (Bottom). The predicted AvrHah1 EBE is in purple, whereas the AvrBs3 EBE has two nucleotide extensions on either side marked in yellow. Similar RVDs between AvrHah1 and AvrBs3 are depicted by green highlighting.
Fig. 6.
Fig. 6.
Tomato gene activation in response to AvrHah1. (A) Semiquantitative RT-PCR in tomato inoculated with the indicated X. gardneri (Xg) strains (24 hpi; OD600 = 0.25); * indicates genes with predicted promoter EBEs for AvrHah1 (e.g., proposed direct targets). (B) Transient luciferase reporter assay in N. benthamiana. Promoter::luciferase constructs are displayed along the x axis. Expression binary vectors carrying AvrHah1 or the bHLH transcription factors are listed on the left, and an “X” signifies codelivery with a promoter::luciferase reporter; * indicates significantly different luciferase activity from promoter background (codelivered with empty Agrobacterium) (P < 0.001).
Fig. 7.
Fig. 7.
dTALEs demonstrate that the bHLH transcription factors and the PL are S genes of AvrHah1. (A) Semiquantitative RT-PCR in tomato infected with XgΔavrHah1 complemented with dTALEs for the bHLH transcription factors (dT bHLH3 and dT bHLH6), the PL (dT PL), and the PE (dT PE) (24 hpi; OD600 = 0.25). (B) Quantitative water-soaking measurements were obtained by centrifugation and weighing of apoplastic fluid from infected tomato leaves (48 hpi; OD600 = 0.1; 20 min water bath). Average weights and SEs from 12 samples (each consisting of two 0.5-cm2 leaf discs) are shown.
See this image and copyright information in PMC

References

    1. Potnis N, et al. Bacterial spot of tomato and pepper: diverse Xanthomonas species with a wide variety of virulence factors posing a worldwide challenge. Mol Plant Pathol. 2015;16(9):907–920. - PMC - PubMed
    1. Ma X, Lewis Ivey ML, Miller SA. First report of Xanthomonas gardneri causing bacterial spot of tomato in Ohio and Michigan. Plant Dis. 2011;95(12):1584. - PubMed
    1. Quezado-Duval AM, Leite RP, Jr, Truffi D, Camargo LEA. Outbreaks of bacterial spot caused by Xanthomonas gardneri on processing tomato in central-west Brazil. Plant Dis. 2004;88(2):157–161. - PubMed
    1. Timilsina S, et al. Multilocus sequence analysis of xanthomonads causing bacterial spot of tomato and pepper plants reveals strains generated by recombination among species and recent global spread of Xanthomonas gardneri. Appl Environ Microbiol. 2015;81(4):1520–1529. - PMC - PubMed
    1. Schornack S, Minsavage GV, Stall RE, Jones JB, Lahaye T. Characterization of AvrHah1, a novel AvrBs3-like effector from Xanthomonas gardneri with virulence and avirulence activity. New Phytol. 2008;179(2):546–556. - PubMed

Publication types

MeSH terms