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Biological control agent Agrobacterium vitis strain ARK-1 suppresses expression of the virD2 and virE2 genes in tumorigenic A. vitis

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Abstract

A nonpathogenic strain of Agrobacterium (=Rhizobium) vitis, ARK-1, limits the development of crown gall of grapevine (Vitis vinifera L.). Co-inoculation of grapevine shoots with ARK-1 and the tumorigenic (Ti) strain VAT03-9 at a 1:1 cell ratio resulted in significantly lower expression of the virulence genes virD2 and virE2 of VAT03-9 1 day after inoculation (dai) compared with expression levels when shoots were inoculated only with VAT03-9. In contrast, nonpathogenic A. vitis strain VAR06-30, which does not limit the development of crown gall of grapevine, co-inoculated with VAT03-9 did not affect expression levels of virD2 and virE2 under the same conditions. ARK-1 began to suppress the VAT03-9 population by seven dai, but no such effect was observed with VAR06-30 during the nine dai study period. Thus, the biological control activity of ARK-1 is likely based on the suppression of essential virulence genes.

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Acknowledgments

This work was supported by a Japan Society for the Promotion of Science KAKENHI Grant (Number 2550038). I thank Associate Prof. Y. Noutoshi (Okayama University Okayama, Japan) for providing useful advice on experimental design.

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Authors and Affiliations

  1. Okayama Prefectural Technology Center for Agriculture, Forestry and Fisheries, Research Institute for Agriculture, 1174-1 Koudaoki, Akaiwa City, Okayama, 709-0801, Japan

    Akira Kawaguchi

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  1. Akira Kawaguchi
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Correspondence to Akira Kawaguchi.

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Fig. S1

Stability of expression of four candidate reference genes in grapevine shoots. Stability values (M) for the four reference genes were evaluated with the geNorm algorithm (http://medgen.ugent.be/~jvdesomp/genorm/) in bacterial cells of the tumorigenic Agrobacterium vitis strain inoculated into shoots of the grapevine plant. In the plot, genes are ranked from the least (left) to the most (right) stable. The pyrG gene had the highest expression stability among the reference genes, and was chosen for use in the present study. However, the pairwise variations (V n /V n+1) did not show values (geNorm V ratio < 0.15) for the optimal number of reference genes (data not shown). (JPEG 80 kb)

Fig. S2

Amplification products of partial sequences of (a) virD2, (b) virE2, and (c) pyrG obtained by RT-qPCR separated by gel electrophoresis. Lanes: M, DNA ladder marker; 1, VAT03-9; 2, ARK-1 plus VAT03-9; 3, VAR06-30 plus VAT03-9; 4, ARK-1; 5, VAR06-30; 6, distilled water. The expected sizes of the RT-PCR products are 61-bp of virD2 (a), 59-bp of virE2 (b), and 69-bp of pyrG (c). (JPEG 170 kb)

Fig. S3

Illustration of the experimental design used to study of the population dynamics during coexistence of the nonpathogenic strains (ARK-1sc or VAR06-30sc) with the tumorigenic strain VAT03-9n in grapevine plants. (a) A 5-μL drop of the mixed cell suspension was dropped onto a needle-prick wound on the grapevine shoot. (b) Shoot samples that included the wound site were collected from 5 plants at each of eight times. (c) After washing and crushing the samples, ten-fold serial dilutions were plated onto St-CuSO4-PSA and nal-PSA media. hai, hours after inoculation; dai, days after inoculation. (JPEG 206 kb)

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Kawaguchi, A. Biological control agent Agrobacterium vitis strain ARK-1 suppresses expression of the virD2 and virE2 genes in tumorigenic A. vitis . Eur J Plant Pathol 143, 789–799 (2015). https://doi.org/10.1007/s10658-015-0730-8

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