Expressed sequence tag (EST) libraries for Metarhizium anisopliae, the causative agent of green muscardine disease, were developed from the broad host-range pathogen Metarhizium anisopliae sf. anisopliae and the specific grasshopper pathogen, M. anisopliae sf. acridum. Approximately 1700 59 end sequences from each subspecies were generated from cDNA libraries representing fungi grown under conditions that maximize secretion of cuticle-degrading enzymes. Both subspecies had ESTs for virtually all pathogenicity-related genes cloned to date from M. anisopliae, but many novel genes encoding potential virulence factors were also tagged. Enzymes with potential targets in the insect host included proteases, chitinases, phospholipases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites. A diverse array of proteases composed 36 % of all M. anisopliae sf. anisopliae ESTs. Eighty percent of the ESTs that could be clustered into functional groups had significant matches (E , 10 25 ) in other ascomycete fungi. These included genes reported to have specific roles in pathogens with plant or vertebrate hosts. Many of the remaining ESTs had their best BLAST match among animal, plant and bacterial sequences. These include genes with plant and microbial counterparts that produce potent antimicrobials. The abundance of transcripts discovered for different functional groups varied between the two subspecies of M. anisopliae in a manner consistent with ecological adaptations of the two pathogens. By hastening gene discovery this project has enhanced development of improved mycoinsecticides. In addition, the M. anisopliae ESTs represent a significant contribution to the extensive database of sequences from ascomycetes that are saprophytes or plant and vertebrate pathogens. Comparative analyses of these sequences is providing important information about the biology and evolutionary history of this clade. INTRODUCTIONAt least 90 genera and more than 700 species of fungi, dispersed in virtually every major taxonomic group except the higher basidiomycetes, have been identified as insect pathogens (Roberts & Humber, 1981). In terms of diversity they rival the plant pathogens, while there are comparatively few (about 40) fungal pathogens of warmblooded animals (Rippon, 1988). Insect-pathogenic fungi such as Metarhizium anisopliae have been extensively studied as key regulatory factors in insect populations and as agents of biocontrol (Inglis et al., 2001). However, attempts to discern the physiological determinants of infection processes and produce a rational plan for strain improvement have often been thwarted by the complexity of fungal responses to host-related signals. Consequently, side effects occurring in a selected or constructed strain are hard to predict and assess, and the full range of engineering possibilities cannot be exploited, due to lack of knowledge of inter-related regulatory and metabolic processes going on in the cell.We need alternative strategies to assess genomes of M. anisoplia...

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Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22°C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton bolls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts.Aspergillus species are associated with disease in plants, insects, man, and other animals (4, 14, 24). For example, Aspergillus flavus causes disease of agronomically important crops, such as corn and peanuts, is second only to Aspergillus fumigatus as the cause of human invasive aspergillosis, and is the Aspergillus species most frequently reported to infect insects.Aspergillus spp. are generally regarded as opportunistic pathogens that require wounds or otherwise weakened hosts for colonization (24). A. flavus, however, also has limited parasitic abilities and, in some cases, can directly invade seeds and colonize living tissues (20). We found previously (35) that Aspergillus spp. produced a much broader spectrum of protein and polysaccharide-hydrolyzing enzymes than did specialized pathogens, which may be indicative of their less-specialized lifestyle. Shieh et al. (29), however, found that aggressiveness toward cotton depended on production of a specific pectinase isoenzyme (P2c) that facilitates spread between cotton boll locules. Weakly pathogenic strains lack P2c, and their growth is limited to individual locules. Likewise, production of proteinases (elastases) in culture has been correlated with isolate virulence to mice and with isolation from human hosts with aspergillosis (12, 13, 25), although soil isolates also produce proteases (17). Proteases may be required for virulence to plants, as resistance to A. flavus in corn kernels derives from a protease inhibitor (5).Populations of A. flavus are highly polymorphic and complex (40), but we do not know if specialized subspecies or physiological races contribute to the broad host range (1, 6, 9). If specializat...

Crop yield is a highly complex quantitative trait. Historically, successful breeding for improved grain yield has led to crop plants with improved source capacity, altered plant architecture, and increased resistance to abiotic and biotic stresses. To date, transgenic approaches towards improving crop grain yield have primarily focused on protecting plants from herbicide, insects, or disease. In contrast, we have focused on identifying genes that, when expressed in soybean, improve the intrinsic ability of the plant to yield more. Through the large scale screening of candidate genes in transgenic soybean, we identified an Arabidopsis thaliana B-box domain gene (AtBBX32) that significantly increases soybean grain yield year after year in multiple transgenic events in multi-location field trials. In order to understand the underlying physiological changes that are associated with increased yield in transgenic soybean, we examined phenotypic differences in two AtBBX32-expressing lines and found increases in plant height and node, flower, pod, and seed number. We propose that these phenotypic changes are likely the result of changes in the timing of reproductive development in transgenic soybean that lead to the increased duration of the pod and seed development period. Consistent with the role of BBX32 in A. thaliana in regulating light signaling, we show that the constitutive expression of AtBBX32 in soybean alters the abundance of a subset of gene transcripts in the early morning hours. In particular, AtBBX32 alters transcript levels of the soybean clock genes GmTOC1 and LHY-CCA1-like2 (GmLCL2). We propose that through the expression of AtBBX32 and modulation of the abundance of circadian clock genes during the transition from dark to light, the timing of critical phases of reproductive development are altered. These findings demonstrate a specific role for AtBBX32 in modulating soybean development, and demonstrate the validity of expressing single genes in crops to deliver increased agricultural productivity.

Ambient pH regulates the expression of virulence genes of Metarhizium anisopliae, but it was unknown if M. anisopliae can regulate ambient pH. Mutants of M. anisopliae altered in production of oxalic acid were evaluated for the interrelationship of ambient pH, buffering capacity added to media, growth, and generation of extracellular proteases and ammonia. Wild-type and acid-overproducing mutants [Acid(M)] grew almost as well at pH 8 as at pH 6, but acid-non-producing [Acid(N)] mutants showed limited growth at pH 8, indicating that acid production is linked to the ability to grow at higher pH. Production of ammonia by M. anisopliae was strongly stimulated by low levels of amino acids in the medium when cells were derepressed for nitrogen and carbon. Likewise, although Aspergillus fumigatus and Neurospora crassa produced some ammonia in minimal media, addition of low levels of amino acids enhanced production. Ammonia production by A. fumigatus, N. crassa and M. anisopliae increased the pH of the medium and allowed production of subtilisin proteases, whose activities are observed only at basic pH. In contrast, protease production by the Acid(M) mutants of M. anisopliae was greatly reduced because of the acidification of the medium. This suggests that alkalinization by ammonia production is adaptive by facilitating the utilization of proteinaceous nutrients. Collectively, the data imply that ammonia may have functions related to regulation of the microenvironment and that it represents a previously unconsidered virulence factor in diverse fungi with the potential to harm tissues and disturb the host's immune system.