SummaryLignification of cell walls during plant development has been identified as the major factor limiting forage digestibility and concomitantly animal productivity. cDNA sequences encoding a key lignin biosynthetic enzyme, cinnamyl alcohol dehydrogenase (CAD), were cloned from the widely grown monocotyledonous forage species tall fescue ( Festuca arundinacea Schreb.). Recombinant tall fescue CAD expressed in E. coli exhibited the highest V max / K m values when coniferaldehyde and sinapaldehyde were used as substrates.Transgenic tall fescue plants carrying either sense or antisense CAD gene constructs were obtained by microprojectile bombardment of single genotype-derived embryogenic suspension cells. Severely reduced levels of mRNA transcripts and significantly reduced CAD enzymatic activities were found in two transgenic plants carrying sense and antisense CAD transgenes, respectively. These CAD down-regulated transgenic lines had significantly decreased lignin content and altered ratios of syringyl (S) to guaiacyl (G), G to p -hydroxyphenyl (H) and S to H units. No significant changes in cellulose, hemicellulose, neutral sugar composition, p -coumaric acid and ferulic acid levels were observed in the transgenic plants. Increases of in vitro dry matter digestibility of 7.2 -9.5% were achieved in the CAD down-regulated lines, thus providing a novel germplasm to be used for the development of grass cultivars with improved forage quality.

This study was conducted to explore fungal endophyte communities inhabiting native switchgrass plants from the tallgrass prairie of northern Oklahoma. The primary focus was to isolate these endophytes in pure culture from surface-sterilized plant tissues and provide taxonomic identifications based on comparative analysis of ITS rDNA gene sequences. From these data, we evaluated the biodiversity of these potentially beneficial endosymbionts from this rapidly disappearing habitat of the Great Plains. While important from a strictly conservationist standpoint, this survey further allowed us to identify candidate endophytes for introduction into commercial switchgrass cultivars for biomass enhancement. A total of 210 whole plant samples were collected at early vegetative, full reproductive and senescence stages. Fungal endophytes were isolated, identified to species level when possible, and grouped into communities based on plant part, collection month and part of the prairie from which the plants were collected. Species diversity for each community was estimated by Shannon diversity index, and differences in diversity indices were compared using a t-test. The presence of fungal species representing at least 18 taxonomic orders suggests a high level of diversity in switchgrass endophyte communities. The fungal communities from shoot tissue had significantly higher species diversity than communities from the root tissue. The abundance of taxa assigned to the order Hypocreales (to which mutualistic, clavicipitaceous endophytes of coolseason grasses belong) found in shoot (64%) and root tissues (39%) throughout the growing season suggests great potential for utilizing these endophytes for enhancing biomass production and stress resistance of this important bioenergy crop.

Dry matter digestibility is one of the most important characteristics of forage. The major constraint on ruminant digestion of forage cell walls is lignin. Sequences of cDNA encoding a key lignin biosynthetic enzyme, caffeic acid O-methyltransferase (COMT), was cloned from the widely grown monocot forage species tall fescue (Festuca arundinacea Schreb.). Enzymatic properties of recombinant COMT protein expressed in E. coli were determined using six substrates. The preferred substrates for tall fescue recombinant COMT were 5-hydroxyferulic acid and caffeoyl aldehyde. Transgenic tall fescue plants carrying either sense or antisense COMT gene constructs were obtained by microprojectile bombardment of single-genotype-derived embryogenic suspension cells. Consistent and closely related molecular and biochemical data demonstrated that two co-suppressed transgenic lines were down-regulated in their lignin biosynthesis. These COMT down-regulated transgenic tall fescue plants showed substantially reduced levels of transcripts, significantly reduced enzymatic activities, significantly decreased lignin content, apparently altered lignin composition and significantly increased (9.8-10.8%) digestibility.

Aspergillus fumigatus is the predominant mold pathogen in immunocompromised patients. In this study, we present the first characterization of the small GTPase RacA in A. fumigatus. To gain insight into the function of racA in the growth and pathogenesis of A. fumigatus, we constructed a strain that lacks a functional racA gene. The ⌬racA strain showed significant morphological defects, including a reduced growth rate and abnormal conidiogenesis on glucose minimal medium. In the ⌬racA strain, apical dominance in the leading hyphae is lost and, instead, multiple axes of polarity emerge. Intriguingly, superoxide production at the hyphal tips was reduced by 25% in the ⌬racA strain. Treatment of wild-type hyphae with diphenylene iodonium, an inhibitor of NADPH oxidase, resulted in phenotypes similar to that of the ⌬racA strain. These data suggest that ⌬racA strain phenotypes may be due to a reduction or alteration in the production of reactive oxygen species. Most surprisingly, despite these developmental and growth abnormalities, the ⌬racA strain retained at least wild-type virulence in both an insect model and two immunologically distinct murine models of invasive pulmonary aspergillosis. These results demonstrate that in vitro growth phenotypes do not always correlate with in vivo virulence and raise intriguing questions about the role of RacA in Aspergillus virulence.

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