2012 Annual Report
1a.Objectives (from AD-416):
To determine which alterations to the lignin biosynthetic pathway affect resistance to fungal pathogens and insect herbivory in sorghum, and thereby allowing for deployment of bioenergy sorghum containing the desired lignin modifications without increasing biotic pressures. To discover the metabolic and signaling changes which affect plant-biotic interactions.
1b.Approach (from AD-416):
Specific Objectives: Examine effects of specific modifications to lignin biosynthesis through bmr loci or transgenic overexpression on feeding of greenbugs (phloem feeder), corn stalk borers and fall army worms (chewing insect). Examine the effects of these lignin modifications on the colonization of fungi causing foliar or stalk diseases, anthracnose (Colletotrichum sublineolum), stalk rot (Fusarium spp.) and charcoal rot (Macrophomina phaseolina). In the relevant lines, investigate the potential underlying mechanisms for significantly reduced insect feeding or fungal colonization relative to wild-type. The overall objective is to systematically link steps in lignin biosynthesis and accumulation of metabolites to changes in gene expression or defense signaling.
Approach: To manipulate lignin, we will utilize 4 bmr loci and transgenic overexpression of 4 genes involved in monoliginol biosynthesis. The bmr loci all result in reduced lignin content, but have distinct effects on lignin composition and phenylpropanoid metabolism. We will overexpress genes encoding enzymes in monoliginol biosynthesis at critical branch points and a putative transcriptional regulator of lignin biosynthesis. Resistance to fungal pathogens will be assessed in the field and through greenhouse inoculation with fungal isolates containing GFP to examine fungal growth and extent of fungal penetration. Insect feeding studies will be performed using isolated leaves from staged plants at a fixed position. Phenolic metabolites will be analyzed by GC-MS in lines exhibiting fungal or insect resistance. Global gene expression will be analyzed using a newly developed Agilent sorghum genome microarray.
Seven expression cassettes containing genes involved in lignin biosynthesis were designed and constructed. The plant transformation experiments are nearly complete. These transgenic plants produced from these experiments will be used to address a critical question on whether modifying lignin synthesis pathways will affect susceptibility of sorghum plants to insect pests and fungal pathogens that cause significant crop losses. In research with non-transgenic plants, brown midrib mutants that are impaired in lignin synthesis were compared to normal plants in insect feeding assays using three sorghum pests: greenbugs, fall army worm and corn ear worms. The greenbug feeding experiment showed that six bmr mutants were more sensitive to greenbugs than normal sorghum (wild-type), bmr2, bmr6 or bmr12. Fall army worms and corn ear worms fed bmr6 and bmr12 stalks performed significantly poorer than the larvae fed wild-type stalks. Susceptibility of the bmr mutants and wild-type to two fungal pathogens, which are the causal agents of sorghum diseases anthracnose and stalk rot were assayed. None of the bmr mutants showed increased susceptibility to anthracnose as compared to wild-type. Likewise, no bmr mutants showed increased susceptibility to the stalk rot pathogen compare to wild-type, and bmr6 and bmr12 showed slight resistance to the pathogen based on reduced disease lesion size relative to the wild-type lesion size. Overall, the bmr lines did not show increased susceptibility to the fungal pathogens or insect pests relative to wild-type, which indicates bmr mutants may be used to reduce lignin concentration without increasing the susceptibility of sorghum to its insect pests or fungal pathogens. The knowledge gained from this research will be valuable in the development and deployment of sorghum and other grasses for bioenergy because lignin is a major research target for bioenergy crop improvement.
ARS PI monitoring activities to evaluate research progress includes: email, on-site visits, monthly meetings, discussion at professional conferences and telephone calls.