1a. Objectives (from AD-416):
1. To develop transgenic lines overexpressing phenylpropanoid biosynthetic enzymes. 2. To determine the effects of modifications to lignin biosynthesis through bmr loci or transgenic overexpression on phloem-feeding and chewing insects of sorghum. 3. To determine the effects these lignin modifications have on fungi causing foliar or stalk diseases in sorghum. 4. To determine the alternations in metabolitic profiles of relevant lines with significantly increased resistance to either insects or fungi. 5. To determine the changes in global gene expression profiles of relevant lines with significantly increased resistance to either insects or fungi.
1b. Approach (from AD-416):
Altering lignin content and composition are targets to improve lignocellulosic biomass for bioenergy conversion processes, the affects of these alterations on plant-biotic interactions are unknown. We hypothesize that specific modification to the lignin biosynthetic pathway will alter the defense responses of host plants to insects and/or fungal pathogens. Our aim is to gain insight on the underlying causes that contribute to the altered defense responses. The sorghum genotypes we will use for the proposed experiments will carry three bmr mutants, which reduce lignin content and alter lignin composition. To increase phenolic compounds and lignin, we will utilize transgenic approaches whereby transgenic sorghum events will be generated that harbor cassettes for the over-expressing of PAL, C3H and CRR, three key enzymes monolignol biosynthesis along with a transcription factor strategy through expression of MYB68, a known regulator of lignin biosynthesis. These transgenic events should increase flux through the phenylpropanoid pathway thereby increasing phenolic subunits available for lignin biosynthesis. However, we are cognizant that these transgenic events may display altered phenylpropanoid metabolism and phenolic profiles, without a concomitant lignin content change. We will use the RTx430 background for these experiments, for which we currently have near-isogenic lines for bmr6 and bmr12.
3. Progress Report:
In the past year, our research efforts focused on evaluation of the responses of lignin-modified sorghum to virulent greenbug biotype E. Lines for the experiment included eight transgenic lines carrying three lignin biosynthesis pathway genes (PAL, CCR, and C3H) and the gene encoding the transcription factor MYB, respectively. Two greenbug-resistant lines were used as resistant controls, while Tx 430 and BTx 623 were used as susceptible checks. The bioassay was carried out using the previously reported method (in our progress report for 2011). The tests were repeated three times in a randomized complete block design. Based on the results obtained from the screening assays, all transgenic sorghum lines exhibited a similar pattern in responding to feeding by greenbugs as damage symptoms developed quickly after co-cultured for 3 days. Most of the seedlings of those lines died or were dying by ten days of post-infestation. It was noted that the level of greenbug damage on the seedlings of those lines was not significantly different from their counterparts. In other words, they showed a level of susceptibility comparable to the susceptible checks at the young seedling stage. Therefore, at present we could not determine whether overexpressing those transgenes in sorghum has any impact on plant response to greenbug infestation until the transgenic lines are well characterized in regard to the expression level of those transgenes in the sorghum plants and the subsequent determination of lignin content or composition in those transgenic lines.