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.

Gene expression profiling of a compatible interaction between Douglas-Fir and the root rot fungal pathogen Phellinus sulphurascens.

Douglas-fir (DF) (Pseudotsuga menziesii) is one of the largest and most economically important coniferous species in western North America. Its productivity is greatly affected by the root rot fungus Phellinus sulphurascens Pilát. Evidence of resistance by DF to fungal root pathogens such as P. sulphurascens has been reported but mechanisms of resistance in this compatible pathosystem are not yet known. To better understand the DF-P. sulphurascens interaction, especially at the molecular level, we selected 12 diverse plant genes already identified as defense-related from a cDNA library constructed using root tissues from P. sulphurascens-infected DF seedlings. Using quantitative reverse-transcriptase polymerase chain reaction on infected DF root samples collected at five different time points after inoculation, we found that P. sulphurascens infection significantly elevated expression of the 12 selected genes. In most cases the highest expression level was recorded within 2 to 3 days after inoculation. The constructed cDNA library, which is enriched with defense-related host genes and a number of fungal genes, will continue to serve as a useful resource for future larger-scale gene discovery and functional research on the P. sulphurascens and DF pathosystem.

A cysteine-rich antimicrobial peptide from Pinus monticola (PmAMP1) confers resistance to multiple fungal pathogens in canola (Brassica napus).

Canola (Brassica napus), an agriculturally important oilseed crop, can be significantly affected by diseases such as sclerotinia stem rot, blackleg, and alternaria black spot resulting in significant loss of crop productivity and quality. Cysteine-rich antimicrobial peptides isolated from plants have emerged as a potential resource for protection of plants against phytopathogens. Here we report the significance of an antimicrobial peptide, PmAMP1, isolated from western white pine (Pinus monticola), in providing canola with resistance against multiple phytopathogenic fungi. The cDNA encoding PmAMP1 was successfully incorporated into the genome of B. napus, and it's in planta expression conferred greater protection against Alternaria brassicae, Leptosphaeria maculans and Sclerotinia sclerotiorum. In vitro experiments with proteins extracted from transgenic canola expressing Pm-AMP1 demonstrated its inhibitory activity by reducing growth of fungal hyphae. In addition, the in vitro synthesized peptide also inhibited the growth of the fungi. These results demonstrate that generating transgenic crops expressing PmAMP1 may be an effective and versatile method to protect susceptible crops against multiple phytopathogens.

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

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.

Genomic organization, induced expression and promoter activity of a resistance gene analog (PmTNL1) in western white pine (Pinus monticola).

Cronartium ribicola causes white pine blister rust (WPBR) in subgenus Strobus. Various genetic and molecular approaches were used to detect white pine genes contributing to host resistance. The molecular role of the NBS-LRR family is highly related to plant immuno-activity against various pathogens and pests. In the present study, genomic organization of a resistance gene analog (RGA), designated as PmTNL1, and its allelic variants were characterized in Pinus monticola. PmTNL1 showed high identity with TIR-NBS-LRR proteins from other plants. qRT-PCR revealed that the PmTNL1 transcript was expressed at low basal levels in different tissues and exhibited similar patterns during compatible and incompatible interactions of P. monticola with C. ribicola at early stages post inoculation. In comparison, PmTNL1 was up-regulated significantly in diseased P. monticola tissues with WPBR symptoms. Expression of the PmTNL1 promoter::GUS fusion gene in transgenic Arabidopsis demonstrated that GUS signal appeared only inside phloem tissues of young seedlings and at hydathodes and branching and organ-connecting points in mature Arabidopsis plants. Similar to the endogenous expression pattern for this gene in pine, GUS activity was up-regulated significantly around vascular tissues locally at pathogen infection sites, but little or no induction was observed in response to abiotic stresses. A DNA marker was developed based on variation of the LRR-coding region, and PmTNL1 was mapped to one genetic linkage group using a pedigree with major dominant gene (Cr2) conferring HR resistance to C. ribicola. These results suggest that PmTNL1 may play an important role in white pine partial resistance against C. ribicola.

Association of a novel Pinus monticola chitinase gene (PmCh4B) with quantitative resistance to Cronartium ribicola.

Multiple families of pathogenesis-related (PR) proteins are believed to contribute to plant quantitative resistance to various pathogens. Along with other host PR proteins, PR3 chitinase is one protein component participating in genetic resistance of western white pine (Pinus monticola) to the white pine blister rust (WPBR) pathogen (Cronartium ribicola). In the present study, we characterized a novel P. monticola class IV chitinase gene (PmCh4B) and further analyzed its nucleotide variations in the open-pollinated seed families of diverse geographical distribution and variable levels of quantitative resistance to C. ribicola infection. PmCh4B showed high haplotype diversity (Hd=0.94) and nucleotide diversity (π=0.00965), similar to those of other conifer genes related to environmental stresses. A low level of intragenic linkage disequilibrium (LD) (but most of the levels with statistical significance) was found within a distance of ≈800 bp. Based on PmCh4B haplotype frequency, moderate to high levels of population structure were observed among P. monticola seed families currently used in breeding programs for WPBR resistance (average FST=0.163, P<0.001). Association analysis revealed that allelic variants and multiple single-nucleotide polymorphisms of PmCh4B were significantly associated with quantitative levels of P. monticola resistance against C. ribicola. This work represents the first association study for quantitative resistance in western white pine pathosystem and provides a potential for marker-assisted selection in white pine breeding.

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.

The superfamily of thaumatin-like proteins: its origin, evolution, and expression towards biological function.

Thaumatin-like proteins (TLPs) are the products of a large, highly complex gene family involved in host defence and a wide range of developmental processes in fungi, plants, and animals. Despite their dramatic diversification in organisms, TLPs appear to have originated in early eukaryotes and share a well-defined TLP domain. Nonetheless, determination of the roles of individual members of the TLP superfamily remains largely undone. This review summarizes recent advances made in elucidating the varied TLP activities related to host resistance to pathogens and other physiological processes. Also discussed is the current state of knowledge on the origins and types of TLPs, regulation of gene expression, and potential biotechnological applications for TLPs.

The CC-NBS-LRR Subfamily in Pinus monticola: Targeted Identification, Gene Expression, and Genetic Linkage with Resistance to Cronartium ribicola.

ABSTRACT To investigate disease resistance gene analogs (RGAs) encoding coiled-coil-nucelotide-binding site-leucine-rich repeats (CC-NBS-LRR) proteins in western white pine, degenerate primers targeting the conserved motifs in the NBS domain were designed to amplify RGAs from genomic DNA and cDNA. Sixty-one distinct RGAs were identified with identities to well-known R proteins of the CC-NBS-LRR subfamily. These RGAs exhibited variation of putative amino acid sequences from 13% to 98%, representing a complex CC-NBS-LRR subfamily. A phylogenetic tree constructed from the amino acid sequence alignment revealed that these 61 RGAs were grouped with other CC-NBS-LRR members from angiosperms, and could be further divided into six classes with an identity threshold of 68%. To map RGAs, RGA polymorphisms and a modified amplified fragment length polymorphism (AFLP) method with incorporated sequences from the NBS domain were used to reveal NBS or NBS-AFLP markers. RGA polymorphism study revealed that three off the identified RGAs were not linked to the Cr2 gene imparting resistance to white pine blister rust. However, the AFLP strategy, using bulk segregant analysis (BSA) and haploid segregation analysis, identified 11 NBS-AFLP markers localized in the Cr2 linkage, the closest two to the gene being 0.41 cM and 1.22 cM away at either side. Eight of these markers showed significant amino acid sequence homologies with RGAs.

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.

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.

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.

Characterization, expression and evolution of two novel subfamilies of Pinus monticola cDNAs encoding pathogenesis-related (PR)-10 proteins.

Proteins of the pathogenesis-related (PR)-10 family are induced in many plants by phytopathogens and environmental stresses. A multi-gene family of PR10 proteins has previously been found in the genome of western white pine (Pinus monticola Dougl. ex D. Don). We isolated two novel subfamilies of PR10 cDNAs (PmPR10-2 and PmPR10-3) from P. monticola that are distinct from other PR10 genes (PmPR10-1.1-1.14) reported from the same species. The PmPR10 proteins are grouped in three subfamilies based on similarity in amino acid sequences. The sequence identities of PmPR10 proteins are much higher among members within a subfamily than among members of different subfamilies (86-99% versus 59-68%). Induction of both PmPR10-2 and PmPR10-3 mRNAs was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in needles in response to wounding treatment. PmPR10-3 was also expressed in needles during cold acclimation in winter. Transcript levels of both PmPR10-2 and PmPR10-3 were less than the detectable levels of constitutive expression in roots, stems and vegetative shoots, whereas PmPR10-1.10 mRNA of subfamily I was expressed at various levels. Phylogenetic analysis showed that PmPR10 and PR10 proteins from other conifers are grouped within one clade that is distinct from that of angiosperm PR10 proteins. In the conifer monophyletic group, PR10 sequences diversify into three distinct clusters. Among these three clusters, some PR10 proteins from single conifer species showed greater divergence distances than sequences from other conifer species, suggesting that, within the conifers, the multi-gene family underwent great diversification during evolution. Based on ratios of nonsynonymous to synonymous nucleotide substitutions (Ka/Ks), we speculate that positive selection resulted in the divergence of PmPR10 subfamilies I and III. Possible mechanisms and significance of PR10 gene evolution are discussed.

Analysis of Proteins of Disease-Free and Didymascella thujina-Infected Leaves of Western Red-Cedar (Thuja plicata).

Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude extracts of disease-free and Didymascella thujina-infected foliage of western red-cedar revealed differences in several protein bands and suggests that distinct proteins of D. thujina origin can be identified by SDS-PAGE.

Identification and characterization of the WRKY transcription factor family in Pinus monticola.

The WRKY gene family represents an ancient and highly complex group of transcription factors involved in signal transduction pathways of numerous plant developmental processes and host defense response. Up to now, most WRKY proteins have been identified in a few angiosperm species. Identification of WRKY genes in a conifer species would facilitate a comprehensive understanding of the evolutionary and function-adaptive process of this superfamily in plants. We performed PCR on genomic DNA to clone WRKY sequences from western white pine (Pinus monticola), one of the most valuable conifer species endangered by white pine blister rust (Cronartium ribicola). In total, 83 P. monticola WRKY (PmWRKY) sequences were identified using degenerate primers targeted to the WRKY domain. A phylogenetic analysis revealed that PmWRKY members fell into four major groups (1, 2a+2b, 2c, and 2d+2e) described in Arabidopsis and rice. Because of high genetic diversity of the PmWRKY family, a modified AFLP method was used to detect DNA polymorphism of this gene family. Polymorphic fragments accounted for 17%-35% of total PCR products in the AFLP profiles. Among them, one WRKY AFLP marker was linked to the major resistance gene (Cr2) against C. ribicola. The results of this study provide basic genomic information for a conifer WRKY gene family, which will pave the way for elucidating gene evolutionary mechanisms in plants and unveiling the precise roles of PmWRKY in conifer development and defense response.

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 proteomics approach to identify proteins differentially expressed in Douglas-fir seedlings infected by Phellinus sulphurascens.

We carried out a comparative proteomic study to explore the molecular mechanisms that underlie the defense response of Douglas-fir (DF, Pseudotsuga menziesii) to laminated root rot, a disease caused by Phellinus sulphurascens. 2-DE was conducted on proteins extracted from roots of laboratory-grown, young DF seedlings inoculated with P. sulphurascens. A total of 1303 proteins was detected in 7 dpi infected and uninfected root samples. Among these 1303 proteins, 277 showed differential expression that was statistically significant (p<0.05). Of these 277 proteins, 74 upregulated and 85 downregulated proteins showed at least a two-fold change from controls. Forty seven upregulated and 23 downregulated proteins were selected to be excised and analyzed using LC-MS/MS followed by peptide matching. Our results indicate that the major proteins differentially expressed in P. sulphurascens-infected DF seedlings include those in the following functional groups: disease/defense (27%), metabolism (16%), transcription factors (11%), signal transduction (10%), secondary metabolism (7%) and energy (4%). A number of additional proteins involved in cell structure (3%) and protein synthesis (3%) were also identified. By providing an initial database of candidate pathogenesis-related proteins for the DF-Phellinus sulphurascens pathosystem the results of this study will enable future detailed investigation of gene expression and function.

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.

Characterization of Pin m III cDNA in western white pine.

Maximum accumulation of Pin m III protein in western white pine (Pinus monticola Dougl. ex D. Don) needles occurred during the winter months. To characterize Pin m III, an expression cDNA library from poly(A)+ mRNA of needles was immunoscreened and the full length cDNA was cloned. An open reading frame of 486 bases encodes a protein of 161 amino acid residues with a molecular mass of 18 kD and a predicted isoelectric point of 5.5. The deduced amino acid sequence had some similarities (37%) with an intracellular pathogenesis-related (PR) protein from garden asparagus (Asparagus officinalis L.) and the major pollen allergen from white birch (Betula verrucosa J. F. Ehrh.), which are members of the ribonuclease-like PR-10 family. Phylogenetic analysis provided circumstantial evidence that Pin m III may be grouped with intracellular PRs from asparagus and potato (Solanum tuberosum L.), while the allergens formed another subgroup. Northern analysis showed that the Pin m III gene was preferentially expressed during cold acclimation with the highest expression in the fall and winter months, preceding the peak of Pin m III protein accumulation. Tissue specificity expression analysis indicated that the gene was strongly expressed in roots and twigs. Higher amounts of the homologous protein (Pin l I) and its transcript accumulated in sugar pine (Pinus lambertiana Dougl.) needles infected with blister rust compared with healthy needles.

Root-specific expression of a western white pine PR10 gene is mediated by different promoter regions in transgenic tobacco.

We report here the isolation and characterization of a novel PR10 gene, PmPR10-1.14, from western white pine (Pinus monticola Dougl. ex. D. Don). The PmPR10-1.14 gene encodes a polypeptide exhibiting high similarity with other members of the PR10 family and corresponds to one of six isoforms immunodetected in the roots of western white pine. Northern blot and western immunoblot analyses showed that expression of the PR10 gene family, including PmPR10-1.14, was detected in vegetative tissues constitutively, but not in developing reproductive organs. RT-PCR with gene-specific primers showed that the transcript of PmPR10-1.14 gene was found only in lateral roots and needles during growth. To study PR10 gene regulation at the cellular level, PmPR10-1.14 promoter was fused to the beta-glucuronidase (GUS) report gene, and analyzed for transient and stable gene expression. The transient expression assays in agroinfiltrated tobacco leaves indicated that the core promoter of PmPR10-1.14 gene resided in the sequence from -101 to +69 relative to the first nucleotide of PR10 cDNA. Furthermore, the promoter region from -311 to -101 acted as an enhancer, and the region from -506 to -311 as a silencer. Fluorometric GUS assays of transgenic tobacco plants demonstrated that the longest promoter of 1675 bp directed GUS expression constitutively at high levels in the roots of mature plants, but expression levels were too low to be detectable in other organs in histochemical assays. Histochemical localization analysis showed that PmPR10-1.14 promoter directed a tissue-specific expression exclusively during the initiation and development of the lateral roots. The distal 5' deletion of the promoter to -311 did not decrease the expression level significantly in the roots, suggesting that the cis-regulatory elements necessary for a high level of gene expression reside in the proximal fragment from -311 to +69. As one striking feature, PmPR10-1.14 promoter contains two copies of direct repeated sequences as long as 281 bp at its distal 5' region. Deletion of one copy (-1326 to -1045) or both copies (-1675 to -1045) of the repeated sequences increased gene expression significantly in leaves and stems, which was regulated developmentally. Further deletion to -820 erased the increased gene expression in leaves and stems. These experiments revealed that the root-specific expression of PmPR10-1.14 gene is mediated by different promoter regions with both negative and positive regulatory mechanisms in transgenic tobacco plants.