Arezoo Zamani

Molecular cloning of a pathogen/wound-inducible PR10 promoter from Pinus monticola and characterization in transgenic Arabidopsis plants.

In Pinus monticola (Dougl. ex D. Don), the class ten pathogenesis-related (PR10) proteins comprise a family of multiple members differentially expressed upon pathogen infection and other environmental stresses. One of them, PmPR10-1.13, is studied here by investigating its transcriptional regulation in transgenic Arabidopsis plants. For functional analyses of the PmPR10-1.13 promoter, a 1,316-bp promoter fragment and three 5′ deletions were translationally fused to the ß-glucuronidase (GUS) reporter gene. The 1,316-bp promoter-driven GUS activity first appeared in hypocotyls and cotyledons in 2- to 3-day-old seedlings. As transgenic plants grew, GUS activity was detected strongly in apical meristems, next in stems and leaves. No GUS activity was detected in roots and in reproductive tissues of flower organs. In adult plants, the PmPR10-1.13 promoter-directed GUS expression was upregulated following pathogen infection and by wounding treatment, which generally mimic the endogenous expression pattern in western white pine. Promoter analysis of 5′ deletions demonstrated that two regions between −1,316 and −930, and between −309 and −100 were responsible for the wound responsiveness. By structural and functional comparisons with PmPR10-1.14 promoter, putative wound-responsive elements were potentially identified in the PmPR10-1.13 promoter. In conclusion, PmPR10-1.13 showed properties of a defence-responsive gene, being transcriptionally upregulated upon biotic and abiotic stresses.

Analysis of the Poplar Phloem Proteome and Its Response to Leaf Wounding

Phloem exudate collected from hybrid poplar (Populus trichocarpa x Populus deltoides) was estimated to have more than 100 proteins, of which 48 were identified using LC-MS/MS. Comparative two-dimensional gel electrophoresis demonstrated that two phloem exudate proteins were significantly (P<0.05) upregulated 24 h after leaf wounding. These were identified as pop3/SP1 and a thaumatin-like protein. This is the first characterization of a phloem proteome from a tree species.

A Class IV Chitinase Is Up-Regulated by Fungal Infection and Abiotic Stresses and Associated with Slow-Canker-Growth Resistance to Cronartium ribicola in Western White Pine (Pinus monticola).

ABSTRACT In the present study, in a candidate gene approach, a class IV chitinase gene (PmCh4A) of pathogenesis-related family three was cloned and characterized in western white pine (Pinus monticola). PmCh4A chitinase expression in the different organs of healthy seedlings was below levels detectable by western immunoblot analysis using an antibody raised against PmCh4A protein. However, a 27-kDa isozyme of PmCh4A accu mulated in both susceptible and slow-canker-growth (SCG) resistant seedlings after infection by Cronartium ribicola. As with fungal infection, the application of a signal chemical (methyl jasmonate) and a protein phosphatase 1 and 2A inhibitor (okadaic acid) increased the PmCh4A protein accumulation. Furthermore, another 26-kDa isozyme was expressed specifically in SCG resistant seedlings, providing a potential tool for marker-assisted selection in forest breeding. Wounding treatment also induced expression of the protein. These data suggest that the class IV chitinase PmCh4A is involved in the defense response of western white pine to infection and abiotic stresses.

Identification and characterization of random amplified polymorphic DNA markers linked to a major gene (Cr2) for resistance to Cronartium ribicola in Pinus monticola

ABSTRACT DNA markers tightly linked to resistance (R) genes provide a very powerful tool for both marker-assisted selection in plant breeding and positional cloning of R genes. In the present study, a linkage of random amplified polymorphic DNA (RAPD) markers to the single dominant gene (Cr2) for resistance to white pine blister rust fungus (Cronartium ribicola) was investigated in western white pine (Pinus monticola). A mapping population of 128 individual megagametophytes was generated from seeds of a heterozygous resistant tree (Cr2/cr2), and the corresponding seedlings of each megagametophyte were subjected to the test of phenotype segregation by inoculation with C. ribicola. Bulked segregant analysis and haploid segregation analysis identified eight robust RAPD markers linked to Cr2. This constitutes the first Cr2 genetic linkage map spanning 84.7 cM with four markers only 3.2 cM from Cr2. One sequence (U256-1385) of these linked markers was significantly similar to the Ty3/gypsy-like long terminal direct repeats retrotransposons. Another marker, U570-843, had no significant similarity to any entry in either GenBank or the loblolly genomics data bank. As presumed that the average physical distance per centimorgan is about 10 Mb in P. monticola, it is probably unrealistic to use these DNA markers for positional cloning of the Cr2 gene.

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.

Expression profiling of a complex thaumatin-like protein family in western white pine

The protein content in the plant apoplast is believed to change dramatically as a result of host defense response upon infection with various pathogens. In this study, six novel thaumatin-like proteins (TLPs) were identified in western white pine (Pinus monticola) needle apoplast by a proteomic strategy using two-dimensional protein electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sequent cDNA cloning found that ten P. monticola TLP genes (PmTLP-L1 to -L6 and -S1 to -S4) were expressed in various tissues. Phylogenetic analysis demonstrated that these PmTLP genes belong to a large, complex, and highly diverse plant TLP family. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) using gene-specific primer pairs showed that each PmTLP gene exhibited a characteristic pattern of mRNA expression based on their unique organ distribution, seasonal regulation, and response to abiotic and biotic stresses. A time-course analysis at the early stages of infection by white pine blister rust pathogen Cronartium ribicola revealed that a coordinated upregulation of multiple PmTLP genes was involved in P. monticola major gene (Cr2) resistance. The structural and expressional differentiations suggest that the PmTLP family may contribute to host defense as well as other mechanism.

Gene Cloning and Tissue Expression Analysis of a PR-5 Thaumatin-Like Protein in Phellinus weirii-Infected Douglas-Fir

ABSTRACT In western North America, Douglas-fir (Pseudotsuga menziesii) is the most economically important conifer species susceptible to laminated root rot caused by Phellinus weirii. While attempting to internally sequence an endochitinase found to be up-regulated in P. weirii-infected Douglas-fir roots, we obtained overlapping peptide fragments showing 28% similarity with a PR-5 thaumatin-like protein (TLP) designated PmTLP (Pm for Pseudotsuga menziesi). A rabbit polyclonal antibody was reared against a synthetic peptide composed of a 29-amino-acid-long, conserved, internal sequence of PmTLP and purified by immunoaffinity. Western immunoblot analysis of infected roots of 24-year-old coastalfir showed significantly higher amounts of PmTLP (P < 0.01) closest to the point of P. weirii inoculation and infection than in uninfected regions of the same root. The antibody was also used to screen for PmTLP in roots of 25-year-old interior Douglas-firs naturally infected with a related pathogen, Armillaria ostoyae, and results showed significantly higher levels of PmTLP in bark tissues adjacent to infection (P < 0.05) than in uninfected tissue. Using polymerase chain reaction (PCR)-based cloning, the cDNA of PmTLP was shown to have a 702-bp open reading frame with a signal peptide cleavage site at 155 bp corresponding to a 29-amino-acid-long residue prior to the start of the N-terminal. Based on the deduced amino acid sequence, the molecular mass of the putative PmTLP was calculated to be 21.0 kDa with an isoelectric point of 3.71. Alignment analysis of PmTLP cDNA with a representative genomic DNA PCR sequence showed presence of one intron of variable size, within the coding region. The induction of PmTLP at the site of root infection and its presence in needle tissue suggests a general role for this protein in adaptation to stress and may be part of an integrated defense response initiated by the host to impede further pathogen spread.

The antigen reactive to an anti-white pine blister rust fungal monoclonal antibody (Mab 7) is a homologue of 70-kDa heat shock proteins (a BiP protein).

The production and characterization of monoclonal antibodies (Mab) to the white pine blis- ter rust (WPBR) fungus (Cronartium ribicola) were described by Ekramoddoullah and Taylor (1996). One of the monoclonal antibodies, Mab 7, detected a major mycelial antigen on blister rust. This mono- clonal antibody did not cross-react with proteins from the white pine host or with proteins from other fun-