Identification, characterization, and expression analyses of class II and IV chitinase genes from Douglas-fir seedlings infected by Phellinus sulphurascens.

Laminated root rot (LRR) disease, caused by the fungus Phellinus sulphurascens, is a major threat to coastal Douglas-fir (DF) (Pseudotsuga menziesii) forests in western North America. Understanding host-pathogen interactions of this pathosystem is essential to manage this important conifer root disease. Our research objectives were to identify DF pathogenesis-related (PR) genes and analyze their expression patterns over the course of infection. We constructed a cDNA library of Phellinus sulphurascens-infected DF seedling roots and sequenced a total of 3,600 random cDNA clones from this library. One of the largest groups of identified genes (203 cDNA clones) matched with chitinase genes reported in other plant species. We identified at least three class II and six class IV chitinase genes from DF seedlings. Quantitative reverse-transcriptase polymerase chain reaction analyses showed significant differential expression patterns locally in root tissues and systemically in needle tissues after fungal invasion. Nonetheless, there was a common trend in gene expression patterns for most of the chitinase genes: an upregulation within 12 h of pathogen inoculation followed by down-regulation within 2 to 3 days postinoculation (dpi), and then further upregulation within 5 to 7 dpi. Western immunoblot data showed differential accumulation of class IV chitinases in Phellinus sulphurascens-infected DF seedlings. Further detailed functional analyses will help us to understand the specific role of DF chitinases in defense against Phellinus sulphurascens infection.

Ultrastructural studies of Phellinus sulphurascens infection of Douglas-fir roots and immunolocalization of host pathogenesis-related proteins.

Interactions between roots of Douglas-fir (DF; Pseudotsuga menziesii) seedlings and the laminated root rot fungus Phellinus sulphurascens were investigated using scanning and transmission electron microscopy and immunogold labelling techniques. Scanning electron micrographs revealed that P. sulphurascens hyphae colonize root surfaces and initiate the penetration of root epidermal tissues by developing appressoria within 2 d postinoculation (dpi). During early colonization, intra- and intercellular fungal hyphae were detected. They efficiently disintegrate cellular components of the host including cell walls and membranes. P. sulphurascens hyphae penetrate host cell walls by forming narrow hyphal tips and a variety of haustoria-like structures which may play important roles in pathogenic interactions. Ovomucoid-WGA (wheat germ agglutinin) conjugated gold particles (10 nm) confirmed the occurrence and location of P. sulphurascens hyphae, while four specific host pathogenesis-related (PR) protein antibodies conjugated with protein A-gold complex (20 nm) showed the localization and abundance of these PR proteins in infected root tissues. A thaumatin-like protein and an endochitinase-like protein were both strongly evident and localized in host cell membranes. A DF-PR10 protein was localized in the cell walls and cytoplasm of host cells while an antimicrobial peptide occurred in host cell walls. A close association of some PR proteins with P. sulphurascens hyphae suggests their potential antifungal activities in DF roots.

Host-Pathogen Interactions in Douglas-Fir Seedlings Infected by Phellinus sulphurascens.

ABSTRACT Several aspects of the host-pathogen interaction between Douglas-fir (Pseudotsuga menziesii) and the fungal pathogen Phellinus sulphurascens were investigated in an in vitro inoculation system using young seedlings and fungal mycelia. Light microscopy confirmed that P. sulphurascens mycelia can successfully penetrate host epidermal cells within 3 days postinoculation (dpi). Extensive fungal colonization and cortical cell decay occurred within 14 dpi. Western immunoblot studies showed significant upregulation (five to sixfold) of four specific pathogenesis-related (PR) proteins in infected roots. These proteins were a Douglas-fir thaumatin-like protein (PmTLP), an endochitinase protein (ECP), a Douglas-fir PR10 (DF-PR10) protein (PsemI), and a 10.6-kDa antimicrobial peptide (PmAMP1). The highest accumulation of PmTLP and PmAMP1 occurred at 12 dpi, whereas accumulations of the ECP and DF-PR10 proteins peaked at 7 dpi. For both inoculated and control Douglas-fir seedlings, only one of the four PR proteins, PmAMP1, was clearly detectable in needles. Immunolocalization experiments using fluorescein isothiocyanate-conjugated secondary antibodies confirmed accumulation of all four PR proteins mainly in and around cell walls of root cortical tissues. Overall, the highest immunofluorescence was observed in infected roots at 12 dpi, whereas labeling in control roots was negligible at all sample times. The ECP produced the highest fluorescence; the DF-PR10 the lowest. Upregulation and localization of these PR proteins in cortical tissues of inoculated roots suggest that they play a defensive role in response to infection by P. sulphurascens. This in vitro inoculation system will facilitate further proteomic and genomic studies of this important pathosystem.

Cloning and Characterization of a Putative Antifungal Peptide Gene (Pm-AMP1) in Pinus monticola.

ABSTRACT We have been working on proteins that are involved in the defense response of western white pine (WWP) (Pinus monitcola) to the blister rust fungus Cronartium ribicola. Our objective was to identify candidate genes that could be used for improving resistance of WWP to this rust pathogen. During proteomic analysis of bark proteins extracted from WWP trees exhibiting slow-canker-growth (SCG) resistance, a 10.6-kDa peptide, termed Pm-AMP1, was found to be enriched at the receding canker margin. The cDNA encoding this peptide was cloned and characterized. A BLASTX search revealed that the Pm-AMP1 encoded by its cDNA has a 50% homology with MiAMP1, a broad-spectrum antifungal protein isolated from Macadamia integrifolia. Based on the deduced amino acid sequence, an antibody was produced against the Pm-AMP1. Immunochemical quantification of the Pm-AMP1 in bark samples of susceptible WWP trees revealed this protein to be barely detectable in the cankered tissues, but occurring in higher concentrations in healthy tissues away from canker margins. Foliage of SCG-resistant trees contained higher concentrations of the Pm-AMP1 than foliage from susceptible cankered trees. Both wounding and methyl jasmonate treatment of WWP needles induced the expression of this protein, further supporting its putative role as a defense response protein.

Isolation, genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine ( Pinus monticola Dougl. ex. D. Don.).

Western white pine ( Pinus monticola Dougl. ex. D. Don., WWP) shows genetic variation in disease resistance to white pine blister rust ( Cronartium ribicola). Most plant disease resistance (R) genes encode proteins that belong to a superfamily with nucleotide-binding site domains (NBS) and C-terminal leucine-rich repeats (LRR). In this work a PCR strategy was used to clone R gene analogs (RGAs) from WWP using oligonucleotide primers based on the conserved sequence motifs in the NBS domain of angiosperm NBS-LRR genes. Sixty-seven NBS sequences were cloned from disease-resistant trees. BLAST searches in GenBank revealed that they shared significant identity to well-characterized R genes from angiosperms, including L and M genes from flax, the tobacco N gene and the soybean gene LM6. Sequence alignments revealed that the RGAs from WWP contained the conserved motifs identified in angiosperm NBS domains, especially those motifs specific for TIR-NBS-LRR proteins. Phylogenic analysis of plant R genes and RGAs indicated that all cloned WWP RGAs can be grouped into one major branch together with well-known R proteins carrying a TIR domain, suggesting they belong to the subfamily of TIR-NBS-LRR genes. In one phylogenic tree, WWP RGAs were further subdivided into fourteen clusters with an amino acid sequence identity threshold of 75%. cDNA cloning and RT-PCR analysis with gene-specific primers demonstrated that members of 10 of the 14 RGA classes were expressed in foliage tissues, suggesting that a large and diverse NBS-LRR gene family may be functional in conifers. These results provide evidence for the hypothesis that conifer RGAs share a common origin with R genes from angiosperms, and some of them may play important roles in defense mechanisms that confer disease resistance in western white pine. Ratios of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) in the WWP NBS domains were greater than 1 or close to 1, indicating that diversifying selection and/or neutral selection operate on the NBS domains of the WWP RGA family.