2013 Annual Report
1a.Objectives (from AD-416):
Identify genes responsible for Brown Stem Rot (BSR) resistance in resistant genotypes.
1b.Approach (from AD-416):
Conduct experiments to silence candidate BSR resistance genes in a resistant genotype. Silenced plants will be infected with BSR and assayed for resistance or susceptibility.
Plant mechanisms for controlling infection by Phialophora gregata, the causal agent of Brown Stem Rot (BSR) in soybeans, are poorly understood. Unlike most soybean-pathogen systems, scoring resistance or susceptibility to BSR takes six to seven weeks. It is unclear if resistance is not induced until that time or if we are unable to detect early resistance by visual inspection. Therefore we are using Virus Induced Gene Silencing (VIGS) and expression analyses to characterize resistance.
We have used VIGS to target three clusters of resistance genes that are located at the Rbs3 locus. Initially, we completed three experiments to evaluate and study the silencing ability of two VIGS constructs (RLPB and RLPC) on BSR resistance. Plants were inoculated with VIGS vectors and constructs when primary leaves were fully expanded (VC stage). Approximately one week later, plants were inoculated with the brown stem rot pathogen (Phialophora gregata, PgA) at the V1 growth stage. The experiments were conducted using relative to an empty vector control (should not cause silencing of Rbs3) and the VIGS gene constructs RLPB and RLPC, which we predicted could silence Rbs3 in BSR resistant variety BSR101. For each round of experiments, we used approximately 55 pots containing 160 plants total. BSR101 was used as the resistant control and a susceptible soybean variety was also used as BSR-susceptible control. Plants inoculated with VIGS vectors developed virus symptoms three to four weeks post-inoculation. Symptoms of BSR began to develop at five weeks post inoculation, and plants were evaluated for foliar and stem symptom severity at six to seven weeks after inoculation. The duration of each experiment was approximately eight to nine weeks from start to finish. No symptoms of BSR were observed in control plants only infected with the VIGS vectors, nor were symptoms of BSR or VIGS observed in non-inoculated plants. However, the BSR inoculations did not result in consistent levels of BSR disease. Severity of BSR disease was low and inconsistent, possibly due to variable temperatures in the greenhouse that suppressed BSR symptom development and perhaps differential suppression by the VIGS constructs. Therefore, we have been focusing on understanding and improving the VIGS protocol, measuring the success of VIGS using an Enzyme-Linked Immunosorbent Assay (ELISA) test, and evaluation of the seed inoculation procedure for use with VIGS and BSR. We have been generating a fair amount of data, but some key results follow. We never detected the Bean Pod Mosaic Virus (BPMV) VIGS vector in 100% of the plants that we inoculated, regardless of variety; therefore this is clearly a factor that has to be accounted for when evaluating the data for potential VIGS. Different soybean varieties seem to have different levels of susceptibility to the BPMV vector, and presumably to VIGS, as measured by different virus titers at various time points after inoculation with the vector. Of those varieties that we have tested, LN12033 seems to be especially susceptible. This also should be considered when planning and evaluating VIGS experiments. Lastly, initial tests done with the seed vascular puncture inoculation procedure (using the engraving tool and needle probes) indicate that this procedure is effective for early inoculation with the BMMV vector. Additional inoculation experiments are in progress.
In order to perform microarray analyses, we need to generate tissue from infected and mock infect plants, resistant and susceptible to BSR. Our research team focused on late responses to BSR. We planted eight pots (three plants per pot) for each of the four treatments. Treatment 1 was BSR101 (resistant) inoculated with type A of P. gregata; treatment 2 was mock-inoculated control with BSR 101; treatment 3 was a susceptible variety inoculated with type A of P. gregata; and treatment 4 was a mock-inoculated control with a susceptible soybean variety. The plants were grown in a growth chamber and the three plants per pot for treatments 1 and 3 were inoculated with P. gregata using a standard stem injection method. Plants were harvested from all four treatments and frozen in liquid nitrogen at four time points: 24 hours post inoculation, six days post-inoculation, 14 days post-inoculation, and 21 days post inoculation. One set of plants was maintained for six to seven weeks post-inoculation to confirm that severe and typical BSR symptoms developed in the inoculated plants. The frozen plant samples were sent on dry ice for completion of the RNA gene expression portion of this study.