Location: Wheat, Sorghum and Forage Research
Project Number: 3042-21220-033-003-I
Project Type: Interagency Reimbursable Agreement
Start Date: Sep 1, 2016
End Date: Aug 31, 2022
Objective:
Stalk pathogens are a serious constraint to biomass production. One of the most important stalk diseases of sorghum is Fusarium stalk rot, caused by several Fusarium species. The fungus can cause extensive internal damage to the stalk, reduce photosynthetic capacity and result in lodging, thus impairing biomass yield and harvest. There is little information available on effects of stalk pathogens on energy sorghums, but it is likely that these diseases negatively impact yield, quality and usability.
We have previously shown that biological conversion has significantly increased efficiency when reduced lignin lines are used, while, for thermal conversion, greater lignin content increases available energy. We pioneered the development of near-isogenic lines with mutations in two genes for enzymes in the lignin biosynthesis pathway, brown midrib (bmr)-6 and bmr12. These near-isogenic lines have served as the basis for work on cell walls, and saccharification and fermentation. Further research with these lines showed that phenolic intermediates accumulate differentially in different lines, affected by the bmr gene and genetic background. The bmr lines are not only significantly reduced in lignin content but bmr can also affect the composition of lignin subunits; lignin is normally composed of three major monolignols. Recently, a third bmr gene, bmr2, has been characterized, although less is known about the effects of this mutant on lignin composition and phenolic accumulation. We currently have crosses involving the three mutant bmr genes bmr2, 6 and 12, which allows for investigating the interaction of the fungus with combinations of these genes. In collaboration with University of Nebraska we have generated sorghum transformed with an upregulated gene for a transcription factor, Myb68, that controls genes involved in the lignin biosynthetic pathway; these transgenic lines have increased lignin content. Analyses of these lines have revealed that they have significantly increased potential energy.
This laboratory has extensive experience with stalk diseases. We have shown that reduced lignin lines are not more susceptible to these pathogens (bmr2) and in some cases are more resistant (bmr6, bmr12), than near-isogenic wild-type (WT) lines, counter to previously-held concepts. This includes the sorghum pathogen Fusarium thapsinum, which produces significantly reduced lengths on bmr plants, as compared with WT. We have also demonstrated that several pathogens are significantly inhibited in growth by the phenolic compound, ferulic acid. However, F. thapsinum is not inhibited in vitro by this compound, suggesting that other factors are involved in the increased resistance observed in bmr lines.
The hypotheses to be tested in this proposal are: lignin composition significantly affects response of sorghum plants to stalk pathogens; changes in metabolites in sorghum modified for lignin content increase resistance to stalk pathogens; and, changes in metabolites as a result in upregulation of genes increases resistance to sorghum stalk pathogens.
Approach:
The research approach will be a combination of whole plant genetics, biochemistry, molecular biology, metabolomics and transcriptomics. The research outline is as follows:
I. Effects of lignin composition in brown midrib lines on pathogen resistance: Mutations in different bmr genes affect lignin composition differentially, dependent on genetic background. In a cross between a bmr2 line and a bmr6 bmr12 double mutant line, we will assess lignin content and lignin composition in each bmr single mutant, double mutant and the triple mutant. We also will assess response to the stalk pathogens F. thapsinum and Macrophomina phaseolina as compared with parents and WT lines. In this way, we will determine if there is an association between lignin composition and resistance to each pathogen.
II. Metabolic profiles in sorghum modified for lignin content: This approach will assess whether changes in metabolic profiles can be detected in WT lines, bmr lines (single, double and triple mutants) and myb68 overexpression lines in response to pathogens. Lines will be inoculated with the two stalk pathogens (F. thapsinum and M. phaseolina) then tissues will be collected at time zero and two times during development of lesions. The metabolic profiles of inoculated and uninoculated plants will be assessed using gas chromatography-mass spectrometry, to determine whether presence or absence of one or more compounds can be associated with reduced pathogen infection.
III. Gene expression during stalk pathogenesis in sorghum modified for lignin content: Two approaches will be applied.
A. Genes previously shown to be involved in defense in sorghum will be assessed during pathogenesis using reverse transcriptase quantitative PCR (RT-qPCR). Plants of bmr lines (single, double and triple mutants), myb68 overexpression lines, and respective WT lines will be inoculated, tissue will be collected, mRNA extracted and RT-qPCR conducted. Comparisons will be made with uninoculated plants at the zero time point, as well as the same two time points during lesion development of inoculated plants.
B. Transcriptome analyses: From results obtained in II and IIIA, lines of interest among those modified in lignin content, along with respective parents and/or WT lines, will be chosen for transcriptome analyses. Plants will be inoculated and tissues will be collected at the zero time point as well as two time points during lesion development; likewise, tissues will be collected from uninoculated plants. Utilizing RNA-seq, we will compare gene expression between the lignin-modified line and their respective WT line at each time point, as well as to uninoculated controls.
