Publications by A. Zamany

Anti-microbial peptide (AMP): nucleotide variation, gene expression, and host resistance in the white pine blister rust (WPBR) pathosystem

Tue, 18 Sep 2012

Pinus monticola antimicrobial peptide (PmAMP1) inhibits growth of Cronartium ribicola and other fungal pathogens. C. ribicola causes white pine blister rust and has resulted in a dramatic reduction of native white pines across North America. Quantitative disease resistance (QDR) is a highly desirable trait screened in breeding programs for durable resistance against C. ribicola. Along with phenotyping on a collection of germplasms, we analyzed PmAMP1 transcript and protein expression and re-sequenced the full-length gene including its promoter region. A mixed linear model was used to identify the association of single nucleotide polymorphisms (SNPs) with accumulated protein and stem QDR levels. Among 16 PmAMP1 SNPs identified in the present study, we found an association of protein levels with 6 SNPs (P < 0.05), including 2 in the 5′-untranslated region (UTR), 3 in the open reading frame (ORF) region with 2 nonsynonymous SNPs, and 1 SNP in the 3′-UTR. Another set of six SNPs was associated with stem QDR levels (P < 0.05), with one localized in the promoter region and the other five in the ORF region with four nonsynonymous changes, suggesting that multiple isoforms may have antifungal activity to differing degrees. Of three common PmAMP1 haplotypes, the trees with haplotype 2 showed high QDR levels with moderate protein abundance while those trees with haplotype 3 exhibited low QDR levels in the susceptible range and the lowest level of protein accumulation. Thus, an association of gene variations with protein abundance and resistance-related traits may facilitate elucidation of physiological contribution of PmAMP1 to host resistance.

Comparative proteomic profiles of Pinus monticola needles during early compatible and incompatible interactions with Cronartium ribicola

Tue, 18 Sep 2012

The proteomic profiles of primary needles from Cr2-resistant and cr2-susceptible Pinus monticola seedlings were analysed post Cronartium ribicola inoculation by 2-DE. One hundred-and-five protein spots exhibiting significant differential expression were identified using LC–MS/MS. Functional classification showed that the most numerous proteins are involved in defence signalling, oxidative burst, metabolic pathways, and other physiological processes. Our results revealed that differential expression of proteins in response to C. ribicola inoculation was genotype- and infection-stage dependent. Responsive proteins in resistant seedlings with incompatible white pine blister rust (WPBR) interaction included such well-characterized proteins as heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, and intermediate factors functioning in the signal transduction pathways triggered by well-known plant R genes, as well as new candidates in plant defence like sugar epimerase, GTP-binding proteins, and chloroplastic ribonucleoproteins. Fewer proteins were regulated in susceptible seedlings; most of them were in common with resistant seedlings and related to photosynthesis among others. Quantitative RT-PCR analysis confirmed HSP- and ROS-related genes played an important role in host defence in response to C. ribicola infection. To the best of our knowledge, this is the first comparative proteomics study on WPBR interactions at the early stages of host defence, which provides a reference proteomic profile for other five-needle pines as well as resistance candidates for further understanding of host resistance in the WPBR pathosystem.

Antifungal activity of a Pinus monticola antimicrobial peptide 1 (Pm-AMP1) and its accumulation in western white pine infected with Cronartium ribicola.

Fri, 12 Aug 2011

Pinus monticola antimicrobial peptide 1 (Pm-AMP1) was expressed and purified from bacterial cell lysate and its identity and purity confirmed by Western blot analysis using the Pm-AMP1 antibody. Application of Pm-AMP1 resulted in visible hyphal growth inhibition of Cronartium ribicola, Phellinus sulphurascens, Ophiostoma montium, and Ophiostoma clavigerum 3–12 days post-treatment. Pm-AMP1 also inhibited spore germination of several other phytopathogenic fungi by 32%–84% 5 days post-treatment. Microscopic examination of C. ribicola hyphae in contact with Pm-AMP1 showed distinct morphological changes. Seven western white pine (Pinus monticola Douglas ex D. Don) families (Nos. 1, 2, 5, 6, 7, 8, 10) showing partial resistance to C. ribicola in the form of bark reaction (BR) were assessed by Western immunoblot for associations between Pm-AMP1 accumulation and family, phenotype, canker number, and virulence of C. ribicola. There was a significant difference (p < 0.001) in mean Pm-AMP1 protein accumulation between families, with higher levels detected in the full-sib BR families (Nos. 1, 2, 5) than the half-sib BR families (Nos. 6, 7). Family 8, previously described as a Mechanism ‘X’ BR family, had the highest number of BR seedlings and displayed high Pm-AMP1 levels, whereas the susceptible family (No. 10) showed the lowest levels (p < 0.05). Family 1 showed a significant association between Pm-AMP1 accumulation and overall seedling health (p < 0.01, R = 0.533), with higher protein levels observed in healthy versus severely infected seedlings. In general, low Pm-AMP1 levels were observed with an increase in the number of cankers per seedling (p < 0.05), and seedlings inoculated with the avirulent source of C. ribicola showed significantly higher Pm-AMP1 levels (p < 0.05) in the majority of BR families. Cis-acting regulatory elements, such as CCAAT binding factors, and an AG-motif binding protein were identified in the Pm-AMP1 promoter region. Multiple polymorphic sites were identified within the 5′ untranslated region and promoter regions. Our results suggest that Pm-AMP1 is involved in the western white pine defense response to fungal infection, as observed by its antifungal activity on C. ribicola and a range of phytopathogens as well as through its association with different indicators of resistance to C. ribicola.

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

Fri, 15 Jan 2010

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.

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

Fri, 24 Mar 2006

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

Wed, 08 Feb 2006

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 fromsWWP trees exhibiting slow-canker-growth (SCG) resistance, a 10.6-k Daspeptide, 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.

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

Tue, 28 Jun 2005

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 prime 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 prime 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.

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)

Tue, 15 Mar 2005

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 accumulated 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.

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

Thu, 02 Dec 2004

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 menziesii). 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 coastal Douglas-fir 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. Additional keywords: ectotrophic mycelium, defense-related protein, mass spectrometry analysis.

Endochitinase activity in the apoplastic fluid of Phellinus weirii-infected Douglas-fir and its association with over wintering and antifreeze activity

Tue, 30 Sep 2003

Extracellular proteins were extracted from Phellinus weirii infected Douglas-fir (Pseudotsuga menziesii var. menziesii) roots and needles to examine endochitinase activity. Chitinases have been associated with the plant's defence response against fungal attack because they hydrolyse chitin, a structural component of fungal cell walls. Protein separation using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western immunoblot analysis using a polyclonal antibody specific to an endochitinase-like protein (ECP) resulted in the detection of up to three polypeptides between 27 and 30 kDa in size. Two-dimensional gel electrophoresis (2-D PAGE) followed by Western immunoblot analysis revealed that the apoplastic fluid contained multiple ECP isoforms with isoelectric points (pIs) ranging from 5.3 to 5.8 and molecular masses of 27-30 kDa. Chitinase activity in needle and root tissues was measured spectrophotometrically using a colorimetric assay. A gel overlay technique using glycol chitin as a substrate for endochitinase was applied to confirm that the ECP antibody detected an enzymatically active protein. The apoplastic fluid collected from P. weirii-infected winter Douglas-fir needles showed anti-freeze activity and seasonal analysis of needle tissue showed some evidence of ECP accumulation in winter months. ECP was distributed systemically throughout the tree. Increased levels of endochitinase activity in the region of P. weirii infection supports a physiological role for ECP in the plant defence response.

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

Fri, 28 Dec 2001

The production and characterization of monoclonal antibodies (Mab) to the white pine blister 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 monoclonal antibody did not cross-react with proteins from the white pine host or with proteins from other fungal species that were known to infect western white pine. As part of our ongoing work on the potential use of Mab 7 as a plantibody for engineering WPBR-resistant white pine, we report here on the cloning and characterization of cDNA encoding the antigen reactive to Mab 7. Five rounds of immunoscreening of a Uni-ZAP expression cDNA library from poly (a) + mRNA of C. ribicola mycelia with Mab 7 led to the identification of three positive clones. One of them was completely sequenced. Sequence analysis indicated an open reading frame of 2010 bases encoding a protein (designated as Cro r II) of 669 amino acid residues with a molecular mass of 72.9 kDa and a predicted isoelectric point of 5.0. Two-dimensional silver-stained gels and 2-D Western analyses detected multiple spots with the same molecular mass and slightly different pI around pH 5.0, suggesting isoforms of Cro r II. A BLAST search of the NCBI database with the deduced Cro r II protein sequence indicated homology with a group of 70-kDa heat shock proteins. Southern blot hybridizations indicated that the C. ribicola genome contained at least one copy of the Cro r II gene. Western immunoblot analyses revealed that the Cro r II protein was present in mycelial culture, in the infected white pine tissues, in the alternate infected Ribes stage, and in the five different spore stages, suggesting a constitutive role for Cro r II in the fungus.

Detection and seasonal expression pattern of a pathogenesis-related protein (PR-10) in Douglas-fir (Pseudotsuga menziesii) tissues.

Thu, 19 Oct 2000

Previously, a PR-10 protein Pin m III was described in western white pine. In this study, primers based on cDNA of Pin m III were utilized to obtain the genomic sequence of a Pin m III homologue - Pse m I - in Douglas-fir. A comparative analysis of a deduced amino acid sequence of Pin m III and Pse m I genes indicated about 80% similarity between the two protein sequences, and a consensus 20 amino-acid sequence located around the p-loop sequence was used to synthesize a peptide of 20 amino acids. An antibody to this synthetic peptide was able to detect the Pse m I protein in Douglas-fir. The anti-Pse m I antibody was used in a western immunoblot to monitor seasonal variation of the Pse m I in Douglas-fir needles and its level was shown to increase with overwintering of Douglas-fir seedlings. However, unlike the Pin m III, there is no indication that the Pse m I is associated with frost hardiness. Analysis of infected Douglas-fir roots showed a possible trend to up-regulation of Pse m I by pathogens such as the laminated root rot fungus, Phellinus weirii. The expression of Pse m I protein in Douglas-fir seedlings is very low compared to the expression of Pin m III protein in western white pine seedlings. In addition, a light-harvesting complex I protein, PSI-F was identified in Douglas-fir by N-terminal amino acid sequencing.

Production of a polyclonal antibody to Phellinus pini and examination of its potential use in diagnostic assays. (Abstract)

Tue, 27 Sep 2011

Production of a polyclonal antibody to Phellinus pini and examination of its potential use in diagnostic assays

Sat, 26 Aug 2000

In order to differentiate among Phellinus pini, Inonotus tomentosus and Inonotus circinatus a polyclonal antibody was raised to a N-terminal part of 25-kDa P. pini-specific protein. The specificity of the polyclonal antibody produced against a synthetic N-terminal peptide of this protein was investigated for diagnostic purposes using Western immunoblot, indirect enzyme-linked immunosorbent assay (ELISA), and inhibition ELISA techniques. The N-terminal synthetic peptide, used as the immunogen, was found to be more than 80% pure by reverse-phase high-performance liquid chromatography. Following immunization, antisera were collected at three different time intervals. The antibody molecules were purified from the crude antisera using immunoaffinity gel chromatography. Following one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis, Western immunoblot analysis showed that the P. pini I polyclonal-antibody detected the immunogen, the 25-kDa protein, in all but one of the P. pini isolates examined, but in none of the isolates of the nontarget species I. tomentosus and I. circinatus. Nevertheless, cross-reactivity was a problem because the P. pini I polyclonal-antibody also recognized bands at other molecular weights in nearly all of the isolates of the other species tested. With the indirect ELISA the P. pini isolates tended to have higher affinity for the polyclonal antibody than the nontarget species, but some cross-reactivity did occur. Inhibition ELISAs, performed over a range of soluble antigen concentrations (1.56-400 ng/100 ml), failed to show a clear distinction between P. pini and the two Inonotus spp. The low level of cross-reactivity observed for I. tomentosus isolate 52 (9%) was also apparent in the indirect ELISA analysis. All three assays indicated that P. pini isolate 41 was the most antigenic. Despite cross-reactivity, the antibody is useful in Western immunoblot for the diagnosis of most P. pini isolates.

Inonotus tomentosus, I. circinatus, and Phellinus pini are distinguished by their protein patterns. (Abstract)

Wed, 28 Sep 2011