Genetic background impacts soluble and cell wall-bound aromatics in brown midrib mutants of sorghum.

Sorghum (Sorghum bicolor (L.). Moench) BMR-6 and BMR-12 encode cinnamylalcohol dehydrogenase and caffeic acid-O-methyltransferase, respectively. We have evaluated the impact of two bmr alleles, bmr-6 and bmr-12, respectively, on soluble and wall-bound aromatics in near isogenic, wild-type (WT), bmr-6, bmr-12 and double-mutant (DM; bmr-6 and bmr-12) plants in two genetic backgrounds, RTx430 and Wheatland. Immunoblots confirmed that COMT protein was essentially absent in bmr-12 and DM plants, but was present in bmr-6 and WT plants. In contrast, although CAD activity was not detected in bmr-6 and DM plants, proteins crossreacting to anti-CAD sera were found in stem extracts from all genotypes. In both sorghum backgrounds, WT plants had lowest amounts of free aromatics, higher levels of cell wall-bound pCA and FA esters and guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) lignins. Soluble aromatics and cell wall phenolic ester content in Wheatland DM plants resembled that of Wheatland bmr-6 plants, whereas in the RTx430 background, levels of these components in the DM plants more closely resembled those observed in bmr-12 plants. In both backgrounds, bmr-6 plants exhibited reduced levels of G, S, and H lignins relative to WT, and increased incorporation of G-indene into lignin. In bmr-12 plants, there was greater incorporation of G- and 5-hydroxyguaiacyl (5-OHG) lignin into cell walls. Histochemical staining of internode sections from Wheatland plants indicated that apparent lignification of cortical sclerenchyma and vascular bundle fibers was greatest and most uniform in WT plants. Relative staining intensity of these tissues was decreased in bmr-6, followed by bmr-12 plants. DM plants exhibited poor staining of cortical sclerenchyma and vascular bundle fibers.

Opportunities and roadblocks in utilizing forages and small grains for liquid fuels.

This review focuses on the potential advantages and disadvantages of forages such as switchgrass (Panicum virgatum), and two small grains: sorghum (Sorghum bicolor), and wheat (Triticum aesitvum), as feedstocks for biofuels. It highlights the synergy provided by applying what is known from forage digestibility and wheat and sorghum starch properties studies to the biofuels sector. Opportunities therefore, exist to improve biofuel qualities in these crops via genetics and agronomics. In contrast to cereal crops, switchgrass still retains tremendous exploitable genetic diversity, and can be specifically improved to fit a particular agronomic, management, and conversion platform. Combined with emerging studies on switchgrass genomics, conversion properties and management, the future for genetic modification of this species through conventional and molecular breeding strategies appear to be bright. The presence of brown-midrib mutations in sorghum that alter cell wall composition by reducing lignin and other attributes indicate that sorghum could serve as an important model species for C(4)-grasses. Utilization of the brown-midrib traits could lead to the development of forage and sweet sorghums as novel biomass crops. Additionally, wheat crop residue, and wheat and sorghum with improved starch content and composition represent alternate biofuel sources. However, the use of wheat starch as a biofuel is unlikely but its value as a model to study starch properties on biofuel yields holds significant promise.

Reaction of Sorghum Lines Genetically Modified for Reduced Lignin Content to Infection by Fusarium and Alternaria spp.

Two genes conferring the brown midrib (bmr) trait had been backcrossed into six elite sorghum lines, resulting in reduced lignin in the bmr lines when compared with the wild-type parent. Seed and leaf tissue from field-grown plants, planted at two locations, were screened for Alternaria spp. and Fusarium spp. on semi-selective media. The results suggest that bmr lines do not have increased susceptibility to colonization by Alternaria spp. However, significantly fewer colonies of Fusarium spp., including Fusarium moniliforme, were recovered from seed of reduced lignin lines from two genetic backgrounds. That the bmr trait in some genetic backgrounds might enable increased resistance to colonization by F. moniliforme was further supported by greenhouse experiments in which peduncles of developing heads were inoculated with F. moniliforme. Mean lesion measurements on bmr lines were significantly lower than those resulting from inoculations on wild-type lines. Analysis of near-isogenic lines revealed that mean lesion lengths on bmr lines were significantly less than those produced on their wild-type counterparts in four of the six genetic backgrounds. These results suggest that reduced lignin lines exhibit, in some cases, increased resistance to Fusarium spp., including F. moniliforme.

Association of Plant Color and Pericarp Color with Colonization of Grain by Members of Fusarium and Alternaria in Near-Isogenic Sorghum Lines.

White sorghum (Sorghum bicolor) grain from tan plants is more desirable for human or animal consumption. Colonization by Fusarium and Alternaria spp. was assessed for near-isogenic lines differing in wound response (purple or tan) and pericarp color (red or white) in field-grown grain and in greenhouse-grown plants. Seeds were screened on a semi-selective medium for Alternaria and Fusarium. Significantly fewer fungal colonies were obtained from tan plants with white seed, and fewer numbers of Alternaria colonies were obtained from white seed, regardless of plant color, from an irrigated field, while there were no differences in fungal composition of seeds grown at a nonirrigated field. Screening of seed from the nonirrigated field on Fusarium semi-selective medium yielded fewer Fusarium isolations from seed grown on purple plants compared with seed from tan plants. When inoculated with Alternaria sp. and F. moniliforme, there can be no differences in lesion lengths on tan/white plants when compared with purple/red plants in most assays; in one assay, tan/white plants had smaller lesion lengths following inoculation with F. moniliforme. These results suggest that plants with white seeds were as resistant as plants with the red pericarp trait to colonization by Alternaria and Fusarium spp. However, the results also suggest that under appropriate environmental conditions seed from tan plants may be more susceptible to Fusarium spp. than seed from purple plants.

Identification of new pisatin demethylase genes (PDA5 and PDA7) in Nectria haematococca and non-Mendelian segregation of pisatin demethylating ability and virulence on pea due to loss of chromosomal elements.

Previous studies have shown that high virulence on pea in Nectria haematococca Mating Population VI is linked to the ability to detoxify the pea phytoalexin, pisatin, via demethylation (Pda). To test this linkage further, a highly virulent Pda(+) isolate (34-18) was used as the recurrent parent in backcrosses to Pda(-) isolates, but most of the progeny were low in virulence on pea, and tetrad analysis gave conflicting ratios for the genetic control of Pda. Southern analysis of 34-18 and progeny showed that 34-18 carries a gene similar to PDA1 (PDA1-2), two new PDA genes, PDA5 and PDA7, and that all three genes can be lost during meiosis. Southern analysis of electrophoretic karyotypes showed that PDA1-2 is on a 1.5-Mb dispensable chromosome in 34-18 and that PDA5 and PDA7 are on a 4.9-Mb chromosome in 34-18 but are found on variably sized chromosomes in progeny. Loss of PDA5 or PDA7 in progeny was not generally associated with morphological phenotypes, except in progeny from some crosses between PDA5 parents. Loss of PDA5 was associated with growth abnormalities in these crosses, suggesting that in some genetic backgrounds at least a portion of the PDA5/PDA7 chromosome is essential for normal growth. All highly virulent progeny had PDA1-2 or a combination of PDA5 and PDA7 while isolates that lacked the three genes were low in virulence, supporting the hypothesis that Pda, or genes linked to PDA genes, are necessary for virulence on pea. However, low virulence isolates with PDA genes were also identified, suggesting that there are pathogenicity genes that can segregate independently of PDA genes.

Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans.

The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Galpha subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Galpha protein Gpal, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpal mutant strains. Physiological studies revealed that the Galpha protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpal and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.

Pisatin demethylase genes are on dispensable chromosomes while genes for pathogenicity on carrot and ripe tomato are on other chromosomes in Nectria haematococca.

Studies on the wide-host-range fungus Nectria haematococca MP VI have shown a linkage between virulence on pea and five of nine PDA genes that encode the ability to detoxify the pea phytoalexin, pisatin. Most of the PDA genes are on chromosomes of approximately 1.6 megabases (Mb) and two of these genes, PDA1-2 and PDA6-1, have been demonstrated to reside on approximately 1.6-Mb chromosomes that can be lost during meiosis. Prior studies also have shown that the dispensable chromosome carrying PDA6-1 contains a gene (MAK1) necessary for maximum virulence on chickpea. The present study evaluated whether the other approximately 1.6-Mb chromosomes that carry PDA genes also are dispensable, their relationship to each other, and whether they contain genes for pathogenicity on hosts other than pea or chickpea. DNA from the PDA1-1 chromosome (associated with virulence on pea) and the PDA6-1 chromosome (associated with virulence on chickpea) were used to probe blots of contour-clamped homogeneous electric field (CHEF) gels of isolates carrying different PDA genes and genetically related Pda- isolates. All of the approximately 1.6-Mb PDA-bearing chromosomes hybridized with both probes, indicating that they share significant similarity. Genetically related Pda-progeny lacked chromosomes of approximately 1.6 Mb and there was no significant hybridization of any chromosomes to the PDA1-1 and PDA6-1 chromosome probes. When isolates carrying different PDA genes and related Pda- isolates were tested for virulence on carrot and ripe tomato, there was no significant difference in lesion sizes produced by Pda+ and Pda- isolates, indicating that genes for pathogenicity on these hosts are not on the PDA-containing chromosomes. These results support the hypothesis that the chromosomes carrying PDA genes are dispensable and carry host-specific virulence genes while genes for pathogenicity on other hosts are carried on other chromosomes.