The roles of the phytotoxin coronatine (COR) and salicylic acid (SA)-mediated defenses in the interaction of Pseudomonas syringae pv. tomato DC3000 and tomato (Solanum lycopersicum) were investigated. Unlike findings reported for Arabidopsis thaliana, DC3000 mutants impaired for production of COR or one of its components, coronafacic acid (CFA) or coronamic acid (CMA), induced distinctly different disease lesion phenotypes in tomato. Tomato plants inoculated with the CFA- CMA- mutant DB29 showed elevated transcript levels of SlICS, which encodes isochorismate synthase, an enzyme involved in SA biosynthesis in S. lycopersicum. Furthermore, expression of genes encoding SA-mediated defense proteins were elevated in DB29-inoculated plants compared with plants inoculated with DC3000, suggesting that COR suppresses SlICS-mediated SA responses. Sequence analysis of SlICS revealed that it encodes a protein that is 55 and 59.6% identical to the A. thaliana ICS-encoded proteins AtICS1 and AtICS2, respectively. Tomato plants silenced for SlICS were hypersusceptible to DC3000 and accumulated lower levels of SA after infection with DC3000 compared with inoculated wild-type tomato plants. Unlike what has been shown for A. thaliana, the COR- mutant DB29 was impaired for persistence in SlICS-silenced tomato plants; thus, COR has additional roles in virulence that are SA independent and important in the latter stages of disease development. In summary, the infection assays, metabolic profiling, and gene expression results described in this study indicate that the intact COR molecule is required for both suppression of SA-mediated defense responses and full disease symptom development in tomato.

SummaryVirus-induced gene silencing (VIGS) is an extremely powerful tool for plant functional genomics. We used Tobacco rattle virus (TRV)-derived VIGS vectors expressed from binary vectors within Agrobacterium to induce RNA silencing in plants. Leaf infiltration is the most common method of agroinoculation used for VIGS but this method has limitations as it is laborious for large-scale screening and some plants are difficult to infiltrate. Here we have developed a novel and simple method of agroinoculation, called 'agrodrench', where soil adjacent to the plant root is drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors. By agrodrench we successfully silenced the expression of phytoene desaturase (PDS), a 20S proteasome subunit (PB7) or Mg-protoporphyrin chelatase (Chl H) encoding genes in Nicotiana benthamiana and in economically important crops such as tomato, pepper, tobacco, potato, and Petunia, all belonging to the Solanaceae family. An important aspect of agrodrench is that it can be used for VIGS in very young seedlings, something not possible by the leaf infiltration method, which usually requires multiple fully expanded leaves for infiltration. We also demonstrated that VIGS functioned to silence target genes in plant roots. The agrodrench method of agroinoculation was more efficient than the leaf infiltration method for VIGS in roots. Agrodrench will facilitate rapid large-scale functional analysis of cDNA libraries and can also be applied to plants that are not currently amenable to VIGS technology by conventional inoculation methods.

Genes encoding pathogenesis-related (PR-) proteins isolated from a cDNA library of Fusarium graminearum-infected wheat spikes of scab-resistant cultivar 'Sumai-3' were transformed into susceptible spring wheat, 'Bobwhite' using a biolistic transformation protocol, with the goal of enhancing levels of resistance against scab. Twenty-four putative transgenic lines expressing either a single PR-protein gene or combinations thereof were regenerated. Transgene expression in a majority of these lines (20) was completely silenced in the T(1) or T(2) generations. Four transgenic wheat lines showed stable inheritance and expression of either a single transgene or transgene combinations up to four generations. One line co-expressing a chitinase and beta-1,3-glucanase gene combination, when bioassayed against scab showed a delay in the spread of the infection (type II resistance) under greenhouse conditions. This line and a second transgenic line expressing a rice thaumatin-like protein gene (tlp) which had moderate resistance to scab in previous greenhouse trials, along with susceptible and resistance checks were evaluated for resistance to scab under field conditions. None of the transgenic lines had resistance to scab in the field under conditions of strong pathogen, suggesting these plants lacked effective resistance to initial infection (type I resistance) under these conditions. As far as is known, this is the first report of field evaluation of transgenic wheat expressing genes for PR-proteins against disease resistance.

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.

Genetic transformation of plant cells by Agrobacterium tumefaciens represents a unique case of trans-kingdom sex requiring the involvement of both bacterial virulence proteins and plant-encoded proteins. We have developed in planta and leaf-disk assays in Nicotiana benthamiana for identifying plant genes involved in Agrobacterium-mediated plant transformation using virus-induced gene silencing (VIGS) as a genomics tool. VIGS was used to validate the role of several genes that are either known or speculated to be involved in Agrobacterium-mediated plant transformation. We showed the involvement of a nodulin-like protein and an alpha-expansin protein (alpha-Exp) during Agrobacterium infection. Our data suggest that alpha-Exp is involved during early events of Agrobacterium-mediated transformation but not required for attaching A. tumefaciens. By employing the combination of the VIGS-mediated forward genetics approach and an in planta tumorigenesis assay, we identified 21 ACG (altered crown gall) genes that, when silenced, produced altered crown gall phenotypes upon infection with a tumorigenic strain of A. tumefaciens. One of the plant genes identified from the screening, Histone H3 (H3), was further characterized for its biological role in Agrobacterium-mediated plant transformation. We provide evidence for the role of H3 in transfer DNA integration. The data presented here suggest that the VIGS-based approach to identify and characterize plant genes involved in genetic transformation of plant cells by A. tumefaciens is simple, rapid, and robust and complements other currently used approaches.