Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens

Abstract

We investigated the effects of salicylic acid (SA) and systemic acquired resistance (SAR) on crown gall disease caused by Agrobacterium tumefaciens. Nicotiana benthamiana plants treated with SA showed decreased susceptibility to Agrobacterium infection. Exogenous application of SA to Agrobacterium cultures decreased its growth, virulence, and attachment to plant cells. Using Agrobacterium whole-genome microarrays, we characterized the direct effects of SA on bacterial gene expression and showed that SA inhibits induction of virulence (vir) genes and the repABC operon, and differentially regulates the expression of many other sets of genes. Using virus-induced gene silencing, we further demonstrate that plant genes involved in SA biosynthesis and signaling are important determinants for Agrobacterium infectivity on plants. Silencing of ICS (isochorismate synthase), NPR1 (nonexpresser of pathogenesis-related gene 1), and SABP2 (SA-binding protein 2) in N. benthamiana enhanced Agrobacterium infection. Moreover, plants treated with benzo-(1,2,3)-thiadiazole-7-carbothioic acid, a potent inducer of SAR, showed reduced disease symptoms. Our data suggest that SA and SAR both play a major role in retarding Agrobacterium infectivity.

Figure 1.

Treatment of plants with SA affects Agrobacterium infectivity. A and B, Quantification of transient transformation in the SA-treated plants. Leaf discs derived from mock- or SA-treated N. benthamiana plants were inoculated with GV2260 (carrying the binary vector pBISN1) and were incubated on CIM. A, The inoculated leaves were collected at 2 and 5 dpi and stained with X-Gluc staining solution. B, GUS activity was measured in GV2260-infected leaf discs at 2, 5, and 10 dpi by recording the fluorescence of 4-methylumbelliferone (4-MU; Jefferson et al., 1987) as described (Anand et al., 2007b). These experiments were repeated at least three times with a minimum of 100 leaf discs. C, SA treatment of N. benthamiana plants result in increased SA levels. SA levels in mock- and SA (5 mm)-treated plants were determined 7 d posttreatment. The bars indicate the ses of the means for three independent biological replicates. D, Transient transformation of NahG-expressing tomato plants. Detached leaves of wild-type tomato plants (‘Moneymaker’) and NahG-overexpressing plants were vacuum infiltrated with the disarmed strain A. tumefaciens GV2260 (carrying the binary vector pBISN1) at a low concentration (1 × 10⁵ cfu). Three days postinfection the leaves were stained with X-Gluc for detecting GUS expression. E, Quantification of transient transformation of NahG-expressing tomato plants. GUS activity of the infected leaves was measured by recording the fluorescence of 4-MU 72 to 96 h postinfection. The data presented are the means with sd values of three independent experiments.

Figure 2.

Effect of SA on Agrobacterium virulence and attachment to plant cells. A, Exogenous application of SA to Agrobacterium attenuates its capacity to incite tumors on leaf discs of N. benthamiana. The leaf disc tumorigenesis assays, as described (Anand et al., 2007a, 2007b), were performed with strain A348 induced with AS in the presence or absence of SA (50 μm). The virulence of A348 treated with SA was attenuated as seen from the reduced number of tumors incited when compared with the tumors produced by strain A348 induced in the SA minus medium. Pictures were taken 4 weeks postinfection. B and C, Agrobacterium attachment assay was performed as described (Anand et al., 2007b) with the disarmed strain A. tumefaciens KAt153 (carrying the binary vector pDSKGFPuv) that was mock or SA treated. Leaf discs derived from N. benthamiana plants were incubated with Agrobacterium and the fluorescent bacteria expressing GFPuv attached to the leaf tissues were visualized, as bacterial colonies, along the cut surfaces after 12 h of cocultivation, using a Leica TCS SP2 AOBS confocal system (left panel: GFP fluorescence; right panel: epifluorescence image). C, Quantification of attached bacteria. SA-treated (50 or 100 μm) agrobacteria that were attached to leaf discs were quantified using serial dilution plating as described (Anand et al., 2007b). Bacterial numbers are mean values for three independent experiments with five replicates each.

Figure 3.

Clustering of the differentially expressed Agrobacterium genes upon treatment with AS and SA. We selected all the genes on the Ti plasmid and a few chromosomal genes that were differentially regulated at 4 and 24 h by AS (A4 and A24) and AS plus SA (A+SA4 and A+SA24) for cluster analyses. Data for selected genes were transformed into log2 and gene tree was generated by hierarchical clustering using TMEV (http://www.tm4.org/mev.html). Color codes represent the differential gene expression values, wherein red and green represent the up- and down-regulation of genes, respectively. The genes highlighted in red were selected for validation by quantitative real-time PCR.

Figure 4.

In planta tumorigenesis and leaf disc transformation assays in the gene-silenced plants of N. benthamiana. A, The in planta tumor assay was performed as described (Anand et al., 2007b) on ICS-, NPR1-, and SABP2-silenced and control (TRV:GFP) N. benthamiana plants. Shoots of control and silenced plants were inoculated with the tumorigenic strain A. tumefaciens A348 and the tumors were photographed 6 weeks after inoculation. B and C, Quantification of stable transformation. Axenic leaf discs derived from control and gene-silenced plants were inoculated with A. tumefaciens A348 and were incubated on hormone-free Murashige and Skoog medium. Four weeks after inoculation, the fresh and dry weights of infected leaves were measured. D and E, Quantification of transient transformation. The leaf discs derived from gene-silenced plants were inoculated with the disarmed strain A. tumefaciens GV2260 (carrying the binary vector pBISN1) and GUS activity was determined as described (Anand et al., 2007b) at 2 and 5 dpi. The experiments were replicated three times with a minimum of 100 leaf discs for each gene-silenced plant and the data indicate the average with se values. Letters indicate significant difference using Fisher's lsd test at P < 0.05.

Figure 5.

Stable and transient transformation assays to characterize the effect of BTH application on Agrobacterium infectivity. A, BTH treatment of N. benthamiana plants did not significantly increase free SA. SA levels in the BTH-treated wild-type and TRV:GFP-inoculated plants were determined at 0 and 72 h post BTH treatment. The bars indicate the ses of the means for three biological replicates. B and C, In planta tumor assay was performed as described (Anand et al., 2007b) on the wild-type N. benthamiana and tomato plants mock or BTH treated (0.1–1 mm). Three days posttreatment, shoots were inoculated with the strain A. tumefaciens A348 and were photographed 6 weeks postinfection. D to G, Quantification of stable and transient transformation. Leaf discs derived from mock- or BTH-treated wild-type, TRV:GFP-inoculated, and ICS-silenced N. benthamiana plants were inoculated with either the strain A. tumefaciens A348 or GV2260 (carrying the binary vector pBISN1) and were incubated on hormone-free Murashige and Skoog medium or CIM, respectively. Four weeks after inoculation, fresh and dry weights of A348-infected leaves were measured (D and E). GUS activity was measured as described (Anand et al., 2007b) in GV2260-infected leaf discs at 2 and 5 dpi (F and G). These experiments were repeated at least three times with a minimum of 100 leaf discs for each plant and the data presented are the mean with se values. Letters indicate significant difference using Fisher's lsd test at P < 0.05.