Gene expression profiling of candidate virulence factors in the laminated root rot pathogen Phellinus sulphurascens

Background

Phellinus sulphurascens is a fungal pathogen that causes laminar root rot in conifers, one of the most damaging root diseases in western North America. Despite its importance as a forest pathogen, this fungus is still poorly studied at the genomic level. An understanding of the molecular events involved in establishment of the disease should help to develop new methods for control of this disease.

Results

We generated over 4600 expressed sequence tags from two cDNA libraries constructed using either mycelia grown on cellophane sheets and exposed to Douglas-fir roots or tissues from P. sulphurascens-infected Douglas-fir roots. A total of 890 unique genes were identified from the two libraries, and functional classification of 636 of these genes was possible using the Functional Catalogue (FunCat) annotation scheme. cDNAs were identified that encoded 79 potential virulence factors, including numerous genes implicated in virulence in a variety of phytopathogenic fungi. Many of these putative virulence factors were also among 82 genes identified as encoding putatively secreted proteins. The expression patterns of 86 selected fungal genes over 7 days of infection of Douglas-fir were examined using real-time PCR, and those significantly up-regulated included rhamnogalacturonan acetylesterase, 1,4-benzoquinone reductase, a cyclophilin, a glucoamylase, 3 hydrophobins, a lipase, a serine carboxypeptidase, a putative Ran-binding protein, and two unknown putatively secreted proteins called 1 J04 and 2 J12. Significantly down-regulated genes included a manganese-superoxide dismutase, two metalloproteases, and an unknown putatively secreted protein called Ps0058.

Conclusions

This first collection of Phellinus sulphurascens EST sequences and its annotation provide an important resource for future research aimed at understanding key virulence factors of this forest pathogen. We examined the expression patterns of numerous fungal genes with potential roles in virulence, and found a collection of functionally diverse genes that are significantly up- or down-regulated during infection of Douglas-fir seedling roots by P. sulphurascens.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-603) contains supplementary material, which is available to authorized users.     

Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors

Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping-off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.     

Alcohol oxidase is a novel pathogenicity factor for Cladosporium fulvum, but aldehyde dehydrogenase is dispensable

Cladosporiumfulvum is a mitosporic ascomycete pathogen of tomato. A study of fungal genes expressed during carbon starvation in vitro identified several genes that were up regulated during growth in planta. These included genes predicted to encode acetaldehyde dehydrogenase (Aldh1) and alcohol oxidase (Aox1). An Aldh1 deletion mutant was constructed. This mutant lacked all detectable ALDH activity, had lost the ability to grow with ethanol as a carbon source, but was unaffected in pathogenicity. Aox1 expression was induced by carbon starvation and during the later stages of infection. The alcohol oxidase enzyme activity has broadly similar properties (Km values, substrate specificity, pH, and heat stability) to yeast enzymes. Antibodies raised to Hansenula polymorpha alcohol oxidase (AOX) detected antigens in Western blots of starved C. fulvum mycelium and infected plant material. Antigen reacting with the antibodies was localized to organelles resembling peroxisomes in starved mycelium and infected plants. Disruption mutants of Aox1 lacked detectable AOX activity and had markedly reduced pathogenicity as assayed by two different measures of fungal growth. These results identify alcohol oxidase as a novel pathogenicity factor and are discussed in relation to peroxisomal metabolism of fungal pathogens during growth in planta.     

Variability of gene expression profiles in human blood and lymphoblastoid cell lines

Background

Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction.

Results

A cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 full-length fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence.

Conclusion

This study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis.     

Potassium and sodium uptake systems in fungi. The transporter diversity of Magnaporthe oryzae

In this study, we report an inventory of the K(+) uptake systems in 62 fungal species for which the complete genome sequences are available. This inventory reveals that three types of K(+) uptake systems, TRK and HAK transporters and ACU ATPases, are widely present in several combinations across fungal species. PAT ATPases are less frequently present and are exceptional in Ascomycota. The genome of Magnaporthe oryzae contains four TRK, one HAK, and two ACU genes. The study of the expression of these genes at high K(+), K(+) starvation, and in infected rice leaves revealed that the expression of four genes, ACU1, ACU2, HAK1, and TRK1 is much lower than that of TRK2, TRK3, and TRK4, except under K(+) starvation. The two ACU ATPases were cloned and functionally identified as high-affinity K(+) or Na(+) uptake systems. These two ATPases endow Saccharomyces cerevisiae with the capacity to grow for several generations in low Na(+) concentrations when K(+) was absent, which produces a dramatic increase of cellular Na(+)/K(+) ratio.     

Analysis of genes expressed during rice-Magnaporthe grisea interactions.

Expressed sequence tag (EST) analysis was applied to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe grisea and fungal genes involved in growth within the host during a compatible interaction. A total of 511 clones was sequenced from a cDNA library constructed from rice leaves (Oryza sativa cv. Nipponbare) infected with M. grisea strain 70-15 to generate 296 nonredundant ESTs. The sequences of 293 clones (57.3%) significantly matched National Center for Biotechnology Information database entries; 221 showed homologies with previously identified plant genes and 72 with fungal genes. Among the genes with assigned functions, 32.8% were associated with metabolism, 29.4% with cell/organism defense or pathogenicity, and 18.4% with gene/protein expression. cDNAs encoding a type I metallothionein (MTs-1) of rice and a homolog of glucose-repressible gene 1 (GRG1) of Neurospora crassa were the most abundant representatives of plant and fungal genes, comprising 2.9 and 1.6% of the total clones, respectively. The expression patterns of 10 ESTs, five each from rice and M. grisea, were analyzed. Five defense-related genes in rice, including four pathogenesis-related genes and MTs-1, were highly expressed during M. grisea infection. Expression of five stress-inducible or pathogenicity-related genes of the fungus, including two hydrophobin genes, was also induced during growth within the host. Further characterization of the genes represented in this study would be an aid in unraveling the mechanisms of pathogenicity of M. grisea and the defense responses of rice.     

A fungal symbiont of plant-roots modulates mycotoxin gene expression in the pathogen Fusarium sambucinum

Fusarium trichothecenes are fungal toxins that cause disease on infected plants and, more importantly, health problems for humans and animals that consume infected fruits or vegetables. Unfortunately, there are few methods for controlling mycotoxin production by fungal pathogens. In this study, we isolated and characterized sixteen Fusarium strains from naturally infected potato plants in the field. Pathogenicity tests were carried out in the greenhouse to evaluate the virulence of the strains on potato plants as well as their trichothecene production capacity, and the most aggressive strain was selected for further studies. This strain, identified as F. sambucinum, was used to determine if trichothecene gene expression was affected by the symbiotic Arbuscular mycorrhizal fungus (AMF) Glomus irregulare. AMF form symbioses with plant roots, in particular by improving their mineral nutrient uptake and protecting plants against soil-borne pathogens. We found that that G. irregulare significantly inhibits F. sambucinum growth. We also found, using RT-PCR assays to assess the relative expression of trichothecene genes, that in the presence of the AMF G. irregulare, F. sambucinum genes TRI5 and TRI6 were up-regulated, while TRI4, TRI13 and TRI101 were down-regulated. We conclude that AMF can modulate mycotoxin gene expression by a plant fungal pathogen. This previously undescribed effect may be an important mechanism for biological control and has fascinating implications for advancing our knowledge of plant-microbe interactions and controlling plant pathogens.     

Identification and assignment of function to the genes of Aspergillus fumigatus expressed at 37 degrees C

Aspergillus fumigatus, a fungal pathogen, causes a spectrum of allergic and invasive disorders. In order to rapidly identify genes of this fungus relevant for pathogenesis and as potential antifungal drug targets, 125 expressed sequence tags (ESTs) were generated from 200 phage clones of a non-normalized cDNA library. Out of a novel 68 ESTs, 45 were assigned putative functions based on the sequence similarity. The identities of some of these genes suggest that they may be involved in pathogenesis or autoimmune reactions. Additional genes were identified that are possible targets for the development of antifungal drugs or that may be of use in diagnosing fungal infections.     

Gene expression profile changes in cotton root and hypocotyl tissues in response to infection with Fusarium oxysporum f. sp. vasinfectum

Microarray analysis of large-scale temporal and tissue-specific plant gene expression changes occurring during a susceptible plant-pathogen interaction revealed different gene expression profile changes in cotton root and hypocotyl tissues. In hypocotyl tissues infected with Fusarium oxysporum f. sp. vasinfectum, increased expression of defense-related genes was observed, whereas few changes in the expression levels of defense-related genes were found in infected root tissues. In infected roots, more plant genes were repressed than were induced, especially at the earlier stages of infection. Although many known cotton defense responses were identified, including induction of pathogenesis-related genes and gossypol biosynthesis genes, potential new defense responses also were identified, such as the biosynthesis of lignans. Many of the stress-related gene responses were common to both tissues. The repression of drought-responsive proteins such as aquaporins in both roots and hypocotyls represents a previously unreported response of a host to pathogen attack that may be specific to vascular wilt diseases. Gene expression results implicated the phytohormones ethylene and auxin in the disease process. Biochemical analysis of hormone level changes supported this observation.     

Expression profile analysis of the polygalacturonase-inhibiting protein genes in rice and their responses to phytohormones and fungal infection

Polygalacturonase-inhibiting proteins (PGIPs) are typically leucine-rich repeat (LRR) proteins that can inhibit the activity of fungal polygalacturonases (PGs). In this study, two new Ospgip genes, named Ospgip6 and Ospgip7 with consensus sequence of ten imperfect LRR motif located on rice chromosomes 8 and 9, were identified using BLAST analysis. Both of them appear to be extracellular glycoproteins. To have a global view of the dynamic gene expression pattern, seven Ospgip genes were first analyzed using the Affymetrix rice genome array data from online resource. All of these seven Ospgip genes showed variable expression patterns among tissues/organs. In order to further investigate the potential function of these Ospgip genes, the responses of Ospgip genes to the treatment of various phytohormones (abscisic acid, brassinosteroid, gibberellic acid, 3-indole acetic acid, jasmonic acid, kinetin, naphthalene acetic acid and salicylic acid) as well as fungal infection were analyzed by real-time PCR using time course array. Generally, all the Ospgip genes were slightly up-regulated in the indica rice cultivar Minghui 63 under GA(3), KT and NAA treatments (except Ospgip2, which was down-regulated under KT treatment). In the japonica rice cultivar Zhonghua 11, Ospgip genes were regulated by most treatments with the response time variability. We also analyzed putative cis-elements in the promoter regions of Ospgip genes. This dataset provided a versatile resource to understand the regulatory network of Ospgip genes during the process of phytohormones treatment and fungal infection in the model monocotyledonous plant, rice, and could aid in the transgenic breeding against rice fungal diseases. KEY MESSAGE: All the seven Ospgip genes showed variable expression patterns in Minghui 63 and their expressions were regulated by different phytohormone treatments or fungal infection in Minghui 63 and Zhonghua 11.     

Expressed sequence tags from the flower pathogen Claviceps purpurea

The ascomycete Claviceps purpurea (ergot) is a biotrophic flower pathogen of rye and other grasses. The deleterious toxic effects of infected rye seeds on humans and grazing animals have been known since the Middle Ages. To gain further insight into the molecular basis of this disease, we generated about 10 000 expressed sequence tags (ESTs)—about 25% originating from axenic fungal culture and about 75% from tissues collected 6–20 days after infection of rye spikes. The pattern of axenic vs. in planta gene expression was compared. About 200 putative plant genes were identified within the in planta library. A high percentage of these were predicted to function in plant defence against the ergot fungus and other pathogens, for example pathogenesis-related proteins. Potential fungal pathogenicity and virulence genes were found via comparison with the pathogen–host interaction database (PHI-base; http://www.phi-base.org ) and with genes known to be highly expressed in the haustoria of the bean rust fungus. Comparative analysis of Claviceps and two other fungal flower pathogens (necrotrophic Fusarium graminearum and biotrophic Ustilago maydis ) highlighted similarities and differences in their lifestyles, for example all three fungi have signalling components and cell wall-degrading enzymes in their arsenal. In summary, the analysis of axenic and in planta ESTs yielded a collection of candidate genes to be evaluated for functional roles in this plant–microbe interaction.     

Nod factors and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root formation in Medicago truncatula via the DMI1/DMI2 signalling pathway

Legumes form two different types of intracellular root symbioses, with fungi and bacteria, resulting in arbuscular mycorrhiza and nitrogen-fixing nodules, respectively. Rhizobial signalling molecules, called Nod factors, play a key role in establishing the rhizobium-legume association and genes have been identified in Medicago truncatula that control a Nod factor signalling pathway leading to nodulation. Three of these genes, the so-called DMI1, DMI2 and DMI3 genes, are also required for formation of mycorrhiza, indicating that the symbiotic pathways activated by both the bacterial and the fungal symbionts share common steps. To analyse possible cross-talk between these pathways we have studied the effect of treatment with Nod factors on mycorrhization in M. truncatula. We show that Nod factors increase mycorrhizal colonization and stimulate lateral root formation. The stimulation of lateral root formation by Nod factors requires both the same structural features of Nod factors and the same plant genes (NFP, DMI1, DMI2, DMI3 and NSP1) that are required for other Nod factor-induced symbiotic responses such as early nodulin gene induction and cortical cell division. A diffusible factor from arbuscular mycorrhizal fungi was also found to stimulate lateral root formation, while three root pathogens did not have the same effect. Lateral root formation induced by fungal signal(s) was found to require the DMI1 and DMI2 genes, but not DMI3. The idea that this diffusible fungal factor might correspond to a previously hypothesized mycorrhizal signal, the 'Myc factor', is discussed.