Faith C. Belanger

Evidence for the thiamine biosynthetic pathway in higher-plant plastids and its developmental regulation

Thiamine or vitamin B-1, is an essential constituent of all cells since it is a cofactor for two enzyme complexes involved in the citric acid cycle, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Thiamine is synthesized by plants, but it is a dietary requirement for humans and other animals. The biosynthetic pathway for thiamine in plants has not been well characterized and none of the enzymes involved have been isolated. Here we report the cloning and characterization of two cDNAs representing members of the maize thi1 gene family encoding an enzyme of the thiamine biosynthetic pathway. This assignment was made based on sequence homology to a yeast thiamine biosynthetic gene and by functional complementation of a yeast strain in which the endogenous gene was inactivated. Using immunoblot analysis, the thi1 gene product was found to be located in a plastid membrane fraction. RNA gel blot analysis of various tissues and developmental stages indicated thi1 expression was differentially regulated in a manner consistent with what is known about thiamine synthesis in plants. This is the first report of cDNAs encoding proteins involved in thiamine biosynthesis for any plant species.

Mutualistic fungal endophytes express a proteinase that is homologous to proteases suspected to be important in fungal pathogenicity.

Many cultivated and wild grass species are hosts to mutualistic fungal endophytes. These associations are ecologically and agronomically significant, yet little is known regarding the physiological aspects of the interaction. In the Poa ampla/Acremonium typhinum interaction, a fungal serine proteinase, At1, is surprisingly abundant and may constitute 1 to 2% of the total leaf-sheath protein. Sequence analysis of cDNA and genomic clones indicates that proteinase At1 is a member of the eukaryotic subtilisin-like protease family. It is homologous to proteases suspected to be virulence factors in fungal pathogens of insects, nematodes, and other fungi. Gel blot analysis of RNA extracted from infected leaf-sheath tissue indicates that the proteinase At1 transcript level is extremely high. RNA gel blots and immunoblots of purified enzymes indicate that similar proteinases are produced by Epichloe festucae and Acremonium lolii, the fungal endophytes infecting Festuca rubra subsp. rubra and Lolium perenne, respectively. Fungal expression of proteinase At1-like enzymes may be a general feature of endophyte infection.

Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host

Infection by pathogenic fungi involves breaching the outer layer of the host by either mechanical or enzymatic means. Subtilisin-like proteinases are considered to be important in the infection process of entomopathogenic, nematophagous, and mycoparasitic fungi. Little is known regarding the expression of such proteinases by plant pathogenic fungi. Magnaporthe poae, a fungal pathogen of Kentucky bluegrass, expressed a subtilisin-like proteinase, proteinase Mp1, in the infected roots. Antibody was produced against the purified enzyme. From immunoblot analysis, expression of the proteinase in infected roots correlated with increasing severity of disease symptoms. Sequence analysis of a genomic clone indicated proteinase Mp1 was homologous to other fungal subtilisin-like proteinases. DNA gel blot analysis indicated proteinase Mp1 was encoded by a small gene family.

Endophytic Fungal β-1,6-Glucanase Expression in the Infected Host Grass

Mutualistic fungal endophytes infect many grass species and often confer benefits to the hosts such as reduced herbivory by insects and animals. The physiological interactions between the endophytes and their hosts have not been well characterized. Fungal-secreted proteins are likely to be important components of the interaction. In the interaction between Poa ampla and the endophyteNeotyphodium sp., a fungal β-1,6-glucanase is secreted into the apoplast, and activity of the enzyme is detectable in endophyte-infected plants. Sequence analysis indicates the β-1,6-glucanase is homologous to enzymes secreted by the mycoparasitic fungi Trichoderma harzianum andTrichoderma virens. DNA gel-blot analysis indicated the β-1,6-glucanase was encoded by a single gene. As a secreted protein, the β-1,6-glucanase may have a nutritional role for the fungus. In culture, β-1,6-glucanase activity was induced in the presence of β-1,6-glucans. From RNA gel blots, similar β-1,6-glucanases were expressed in tall fescue (Festuca arundinacea Schreb.) and Chewings fescue (Festuca rubra L. subsp.fallax [Thuill] Nyman) infected with the endophyte species Neotyphodium coenophialum andEpichloë festucae, respectively.

Purification and Characterization of an Endophytic Fungal Proteinase That Is Abundantly Expressed in the Infected Host Grass.

A novel Acremonium typhinum proteinase that is expressed during endophytic infection of the grass Poa ampla Merr. was purified from endophyte-infected leaf sheath tissue. It is a thiol-containing serine alkaline endoproteinase with bound carbohydrate. In the infected host tissue, this proteinase is an abundant protein localized within fungal membrane vesicles and in the plant and/or fungal cell walls. This proteinase was not expressed constitutively during fungus culture. Rather, its expression appeared to be induced by nutrient depletion. Expression of an antigenically similar proteinase was detected in five other endophyte-infected Poa species. The regulated expression of the proteinase in culture and its abundance in infected plant tissue suggest that its expression may be involved in the symbiotic interaction of the plant and the fungus.

Transgenic creeping bentgrass with delayed dollar spot symptoms

Creeping bentgrass (Agrostis palustris Huds) is animportant turfgrass used on golf course greens and fairways. It is susceptibleto a number of fungal pathogens and requires considerable fungicide use fordisease control. Transgenic approaches may be useful in improving the level ofdisease resistance. We have generated transgenic creeping bentgrass plantsexpressing PR5K from Arabidopsis thaliana (L.) Henyh. PR5Kis a receptor protein kinase whose extracellular domain is homologous to thePR5family of pathogenesis-related proteins. In a field test of plants inoculatedwith the fungal pathogen dollar spot (Sclerotiniahomoeocarpa F.T. Bennett) four of the eight transgenic lines showeddelays in disease expression of 29 to 45 days, relative to the control plants.

Mechanism and rate of sugar uptake by Acremonium typhinum, an endophytic fungus infecting Festuca rubra: Evidence for presence of a cell wall invertase in endophytic fungi

Fungal endophytes of the genus Acremonium infect many grass species. They exist in the intercel? lular spaces of the aerial plant parts, most abundantly in the leaf sheaths. They therefore must obtain all of their nutrients from the apoplastic spaces in the plant. This report is the first comprehensive, biochemically based study of sugar uptake by Acremonium spp. The endophytes (Acremonium typhinum) were isolated from Festuca rubra plants and were grown in culture. Indi? vidual colonies were incubated in the presence of (U- 14C)radiolabeled glucose, fructose, or sucrose for 20 minutes and the uptake quantified by scintillation counting. The uptake rates for all three sugars were biphasic, indicating two distinct mechanisms of trans- port. At low sugar concentrations (below 10 mM) the uptake was saturable and had the characteristics of a carrier-mediated mechanism. At high sugar concen? trations (10-100 mM) the uptake was linear suggesting diffusion as the predominant mechanism of uptake. From competition experiments (uptake of labeled sug? ar in the presence of a different unlabeled sugar) it appeared that there were separate carriers for glucose and fructose. Two mechanisms for sucrose uptake were detected, a sucrose carrier and hexose uptake as a result of cell wall invertase. Cell wall invertase activity of the fungal isolates was inducible by growing the fungi on sucrose containing media.

Identification of heat stress-responsive genes in heat-adapted thermal Agrostis scabra by suppression subtractive hybridization

To gain insights into molecular mechanisms of grass tolerance to heat stress, we constructed a suppression subtractive cDNA library to identify heat-responsive genes for a C(3) grass species, thermal Agrostis scabra adapted to heat stress in geothermal areas in Yellowstone National Park. Plants were exposed to 20 degrees C (control) or 35 degrees C for 12d. The SSH analysis was performed with control samples as the driver and heat-stressed samples as the tester. Differentially expressed cDNA fragments were cloned to screen the heat up-regulated library. The SSH analysis identified 120 non-redundant putative heat-responsive cDNAs out of 1180 clones. Genes with homology to known proteins were categorized into six functional groups, with the largest group of genes involved in stress/defense, followed by the group of genes related to protein metabolism. Immunoblot analysis confirmed increases in transcripts of selected genes under heat stress. Transcripts of seven and eight genes were strongly enhanced or induced in shoots and roots, respectively, while two genes were only induced in roots under heat stress. The heat up-regulated genes in thermal A. scabra adapted to long-term heat stress are potential candidate genes for engineering stress-tolerant grasses and for revealing molecular mechanisms of grass adaptation to heat stress.

Expression of pokeweed antiviral proteins in creeping bentgrass

Abstract. Fungal diseases of creeping bentgrass, an important amenity grass used extensively on golf courses, are a serious problem in golf course management. Transgenic approaches to improving disease resistance to fungal diseases are being explored in many species, and in some cases ribosome-inactivating proteins have been found to be effective. We have generated transgenic creeping bentgrass plants expressing three forms of ribosome-inactivating proteins from pokeweed, which are termed pokeweed antiviral proteins (PAP). PAP-Y and PAP-C are nontoxic mutants of PAP; PAPII is the native form of another ribosome-inactivating protein from pokeweed. In creeping bentgrass, PAP-C transformants did not accumulate the protein, suggesting that it is unstable, and in a field test these plants were not protected from infection by the fungal pathogen Sclerotinia homoeocarpa, the causal agent of dollar spot disease. PAPII transformants could accumulate stable levels of the protein but had symptoms of toxicity; one low-expressing line exhibited good disease resistance. PAP-Y transformants accumulated stable levels of protein, and under greenhouse conditions they appeared to be phenotypically normal.

Horizontal gene transfer of a bacterial insect toxin gene into the Epichloë fungal symbionts of grasses

Horizontal gene transfer is recognized as an important factor in genome evolution, particularly when the newly acquired gene confers a new capability to the recipient species. We identified a gene similar to the makes caterpillars floppy (mcf1 and mcf2) insect toxin genes in Photorhabdus, bacterial symbionts of nematodes, in the genomes of the Epichloë fungi, which are intercellular symbionts of grasses. Infection by Epichloë spp. often confers insect resistance to the grass hosts, largely due to the production of fungal alkaloids. A mcf-like gene is present in all of the Epichloë genome sequences currently available but in no other fungal genomes. This suggests the Epichloë genes were derived from a single lineage-specific HGT event. Molecular dating was used to estimate the time of the HGT event at between 7.2 and 58.8 million years ago. The mcf-like coding sequence from Epichloë typhina subsp. poae was cloned and expressed in Escherichia coli. E. coli cells expressing the Mcf protein were toxic to black cutworms (Agrotis ipsilon), whereas E. coli cells containing the vector only were non-toxic. These results suggest that the Epichloë mcf-like genes may be a component, in addition to the fungal alkaloids, of the insect resistance observed in Epichloë-infected grasses.