Ralph A. Dean

The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection cell, an appressorium, that is required for infection of its host. Previously, cAMP was implicated in the endogenous signaling pathway leading to appressorium formation. To obtain direct evidence for the role of cAMP in appressorium formation, the gene encoding the catalytic subunit of the cAMP-dependent protein kinase (cpkA) was cloned, sequenced, and disrupted. Polymerase chain reaction primers designed after highly conserved regions in the same gene from other organisms were used to amplify genomic DNA fragments. The cloned amplification products were used to identify genomic clones. DNA blot analysis indicated that cpkA is present as a single copy in the genome. cpkA consists of 1894 bp, including three short introns sufficient to encode a protein of 539 amino acids with a predicted molecular mass of 60.7 kD. The deduced peptide shares > 45% identity with other catalytic subunits and contains all functional motifs and residues with the addition of a glutamine-rich region at the N terminus. Two transformants, L5 and T-182, in which cpkA had been replaced with a hygromycin resistance gene cassette, were unable to produce appressoria, could not be induced to form appressoria by cAMP, and were nonpathogenic on susceptible rice, even when leaves were abraded. These results were confirmed by analysis of 57 progeny from a cross between transformant L5 and the wild-type laboratory strain 70-6. Other aspects of growth and development, including vegetative growth as well as asexual and sexual competence, were unaffected when measured in vitro. These results provide direct evidence that the cAMP-dependent protein kinase is necessary for infection-related morphogenesis and pathogenesis in a phytopathogenic fungus.

cAMP Regulates Infection Structure Formation in the Plant Pathogenic Fungus Magnaporthe grisea.

Magnaporthe grisea, the causal agent of rice blast, is one of the most destructive fungal pathogens of rice throughout the world. Infection of rice by M. grisea requires the formation of an appressorium, a darkly pigmented, dome-shaped structure. The germ tube tip differentiates into an appressorium following germination of conidia on a leaf surface. When conidia germinate on growth medium or other noninductive surfaces, the emerging germ tube does not differentiate and continues to grow vegetatively. Little is known about the endogenous or exogenous signals controlling the developmental process of infection structure formation. We show here that a hydrophobic surface was sufficient for the induction of the appressorium. Furthermore, we demonstrate that the addition of cAMP, its analogs (8-bromo cAMP and N6-monobutyryl cAMP), or 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) to germinating conidia or to vegetative hyphae induced appressorium formation on noninductive surfaces. The identification of cAMP as a mediator of infection structure formation provides a clue to the regulation of this developmental process. Elucidation of the mechanism involved is not only of biological interest but may also provide the basis for new disease control strategies.

The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection structure called an appressorium that is crucial for host plant penetration. Previously, it was found that cAMP regulates appressorium formation. To further understand the cellular mechanisms involved in appressorium formation, we have cloned a gene (MAC1) encoding adenylate cyclase, a membrane-bound enzyme that catalyzes the production of cAMP from ATP, by using a polymerase chain reaction-based strategy. The entire gene was isolated and subcloned from a large insert bacterial artificial chromosome library. Sequence characterization showed that MAC1 has a high degree of identity with other adenylate cyclase genes from several filamentous fungi as well as yeasts. Gene deletion resulted in reduced vegetative growth, conidiation, and conidial germination. Transformants lacking MAC1 were unable to form appressoria on an inductive surface and were unable to penetrate susceptible rice leaves. mac1- transformants were also sterile and produced no perithecia. Appressorium formation was restored in the presence of exogenous cAMP derivatives. These results confirm that cell signaling involving cAMP plays a central role in the development and pathogenicity of M. grisea.

A genetic map of melon (Cucumis melo L.) based on amplified fragment length polymorphism (AFLP) markers

Abstractu2002Genetic maps facilitate the study of genome structure and evolution, and the identification of monogenic traits or Mendelian components of quantitative traits. We evaluated 228 RAPD, microsatellite and AFLP markers for linkage analysis in melon (Cucumis melo L.) varieties MR-1 (resistant to Fusarium wilt, powdery and downy mildews) and Ananas Yokneum (AY; susceptible to these diseases) and constructed a detailed genetic map. The mapping population consisted of 66 backcross progenies derived from AY×(MR-1×AY). Despite a relatively low level of polymorphism in the species, AFLP markers were found to be more efficient in mapping the melon genome than RAPD or microsatellite markers. The map contains 197 AFLPs, six RAPDs and one microsatellite marker assigned to 14 major and six minor linkage groups, and covers 1942u2005cM with the average distance between adjacent markers of approximately 10u2005cM. The maximum distance allowed between markers is 27.5u2005cM. About 11% of the intervals (20 out of 173) are over 20u2005cM (but less than 27.5u2005cM). The map has immediate utility for identifying markers linked to disease resistance genes that are suitable for marker-assisted breeding. The use of microsatellite markers for integration with other maps is also discussed.

Genes expressed during early stages of rice infection with the rice blast fungus Magnaporthe grisea.

summary A system-wide approach was adopted to further elucidate mechanisms regulating disease outcome between rice and the fungal pathogen Magnaporthe grisea. First, a cDNA library was constructed from M. grisea infected rice at 48 h post-inoculation. The 5 end-sequencing of 619 randomly selected clones revealed 359 expressed sequence tags (ESTs) that had not previously been described. A total of 124 from 260 ESTs with high and moderate similarity scores, based on BlastX, were organized into categories according to their putative function. The largest category of sequences (21%) contained stress or defence response genes. Eleven per cent of identified ESTs were redundant. In a second approach, differential hybridization analysis of the cDNA library using high-density filters resulted in the identification of novel genes and previously characterized M. grisea genes, including several that had previously been implicated in the infection process. A survey of up-regulated cDNA clones revealed clone 29003, which corresponded to the rice peroxidase POX22.3. This gene is known to be expressed in rice upon infection with Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen. Importantly, this approach demonstrates the utility of gene discovery, through ESTs, for revealing novel genes in addition to those previously characterized as being potentially implicated in host-pathogen interactions.

Sorghum Expressed Sequence Tags Identify Signature Genes for Drought, Pathogenesis, and Skotomorphogenesis from a Milestone Set of 16,801 Unique Transcripts

Improved knowledge of the sorghum transcriptome will enhance basic understanding of how plants respond to stresses and serve as a source of genes of value to agriculture. Toward this goal, Sorghum bicolor L. Moench cDNA libraries were prepared from light- and dark-grown seedlings, drought-stressed plants, Colletotrichum-infected seedlings and plants, ovaries, embryos, and immature panicles. Other libraries were prepared with meristems from Sorghum propinquum (Kunth) Hitchc. that had been photoperiodically induced to flower, and with rhizomes from S. propinquum and johnsongrass (Sorghum halepense L. Pers.). A total of 117,682 expressed sequence tags (ESTs) were obtained representing both 3′ and 5′ sequences from about half that number of cDNA clones. A total of 16,801 unique transcripts, representing tentative UniScripts (TUs), were identified from 55,783 3′ ESTs. Of these TUs, 9,032 are represented by two or more ESTs. Collectively, these libraries were predicted to contain a total of approximately 31,000 TUs. Individual libraries, however, were predicted to contain no more than about 6,000 to 9,000, with the exception of light-grown seedlings, which yielded an estimate of close to 13,000. In addition, each library exhibits about the same level of complexity with respect to both the number of TUs preferentially expressed in that library and the frequency with which two or more ESTs is found in only that library. These results indicate that the sorghum genome is expressed in highly selective fashion in the individual organs and in response to the environmental conditions surveyed here. Close to 2,000 differentially expressed TUs were identified among the cDNA libraries examined, of which 775 were differentially expressed at a confidence level of 98%. From these 775 TUs, signature genes were identified defining drought, Colletotrichum infection, skotomorphogenesis (etiolation), ovary, immature panicle, and embryo.

Cloning and characterization of a novel polygalacturonase-encoding gene from Aspergillus parasiticus

Pectinases produced by Aspergillus flavus and A. parasiticus are believed to play a significant role in the ability of these fungi to spread in cotton bolls and other crops. Utilizing a DNA probe, generated by PCR, of the Aspergillus niger pgaII gene, we have isolated a novel, constitutively expressed polygalacturonase (PG)-encoding gene (pecA) from an A. parasiticus cDNA library. DNA sequence analysis and the deduced amino acid (aa) sequence of pecA demonstrated significant identity at the nucleotide and aa levels with other PG of fungal origin. Northern blot analysis of RNA isolated from A. parasiticus grown on either glucose or pectin as the sole carbon source showed that pecA was expressed during growth in both media.

Genetic mapping of a fusarium wilt resistance gene (Fom-2) in melon (Cucumis melo L.)

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis is one of the most devastating diseases in melon production worldwide. The most effective control measure available is the use of resistant varieties. Identifying molecular markers linked to resistance genes can serve as a valuable tool for the selection of resistant genotypes. Bulked segregant analysis was used to identify markers linked to the Fom-2 genes, which confers resistance to races 0 and 1 of the fungal pathogen. Pooled DNA from homozygous resistant or homozygous susceptible progeny of F2 cross between MR-1 and AY was screened using 240xa0PstI/MseI and 200xa0EcoRI/MseI primer combinations to identify AFLP markers linked to Fom-2. Fifteen markers potentially linked to Fom-2 were identified, all with EcoRI/MseI primer pairs. These were mapped relative to Fom-2 in a backcross (BC) population of 60 progeny derived from MR-1 × AY with AY as recurrent parent. Two AFLP markers (ACT/CAT1 and AAC/CAT1) flanked the gene at 1.7 and 3.3xa0cM, respectively. Moreover, AFLP marker AGG/CCC and the previously identified RAPD marker 596-1 cosegregated with Fom-2. These two dominant markers were converted to co-dominant markers by designing specific PCR primers that produced product length polymorphisms between the parents. A survey of 45 melon genotypes from diverse geographic origins with the co-dominant markers demonstrated a high correlation between fragment size and the resistance phenotype. These markers may therefore be useful in marker-assisted breeding programs.

Sequence analysis of the Aspergillus nidulans pectate lyase pelA gene and evidence for binding of promoter regions to CREA, a regulator of carbon catabolite repression

The nucleic acid and deduced amino-acid sequences of the pectate lyase gene (pelA) from Aspergillus nidulans are presented. The pelA gene contains two short introns, 68 and 49 bp in length, and encodes a peptide of 326 amino acids. Five transcriptional start sites are clustered between 65 and 79 bp upstream of the start codon as determined by primer extension. Comparison of the aminoacid sequences of pectate or pectin lyases from bacteria, fungi and plants revealed less than 30% overall identity. However, five regions within these enzymes, in particular domains associated with the active site, are highly conserved with amino-acid similarities greater than 50%. Phylogenetic analysis using the principle of parsimony (PAUP 3.1.1) showed that pelA is most closely related to pectate lyases from plants rather than pectin lyases from other fungi. Previously, pelA was shown to be induced by polygalacturonic acid and repressed in the presence of preferred carbon sources, such as glucose. Gel mobility shift analysis indicates that a PstI-SphI fragment from the pelA promoter binds to a fusion protein composed of the N-terminal part of CREA, a protein involved in carbon catabolite repression, and glutathione-S-transferase. This result suggests CREA may contribute to the regulation of pelA expression.

Latent infection of peach caused by Colletotrichum gloeosporioides and Colletotrichum acutatum

Attached, immature peach fruits were mist-inoculated in the field with isolates of Colletotrichum gloeosporioides or Colletotrichum acutatum, beginning approximately 2 weeks before pit hardening and at regular intervals throughout the growing season until harvest. Immature fruits inoculated with either species initially remained symptomless or developed small, necrotic lesions that did not enlarge. Treatment with paraquat revealed the frequent presence of latent infections. Infections were not found in the noninoculated controls. Up to 100% of fruits inoculated with C. gloeosporioides developed anthracnose symptoms when mature. Inoculation with C. acutatum resulted in up tc 83% of the fruits developing symptoms at maturity. Inoculation date did not influence symptom development. Peach fruit may be infected with either C. acutatum or C. gloeosporioides early in development and remain symptomless until maturity.