Jun-Jun Liu

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.

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.

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.

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.

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.

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.

Western white pine SNP discovery and high-throughput genotyping for breeding and conservation applications

BackgroundWestern white pine (WWP, Pinus monticola Douglas ex D. Don) is of high interest in forest breeding and conservation because of its high susceptibility to the invasive disease white pine blister rust (WPBR, caused by the fungus Cronartium ribicola J. C. Fisch). However, WWP lacks genomic resource development and is evolutionarily far away from plants with available draft genome sequences. Here we report a single nucleotide polymorphism (SNP) study by bulked segregation-based RNA-Seq analysis.ResultsA collection of resistance germplasm was used for construction of cDNA libraries and SNP genotyping. Approximately 36–89 million 2 × 100-bp reads were obtained per library and de-novo assembly generated the first shoot-tip reference transcriptome containing a total of 54,661 unique transcripts. Bioinformatic SNP detection identified >100,000 high quality SNPs in three expressed candidate gene groups: Pinus highly conserved genes (HCGs), differential expressed genes (DEGs) in plant defense response, and resistance gene analogs (RGAs). To estimate efficiency of in-silico SNP discovery, genotyping assay was developed by using Sequenom iPlex and it unveiled SNP success rates from 40.1% to 61.1%. SNP clustering analyses consistently revealed distinct populations, each composed of multiple full-sib seed families by parentage assignment in the WWP germplasm collection. Linkage disequilibrium (LD) analysis identified six genes in significant association with major gene (Cr2) resistance, including three RGAs (two NBS-LRR genes and one receptor-like protein kinase -RLK gene), two HCGs, and one DEG. At least one SNP locus provided an excellent marker for Cr2 selection across P. monticola populations.ConclusionsThe WWP shoot tip transcriptome and those validated SNP markers provide novel genomic resources for genetic, evolutionary and ecological studies. SNP loci of those candidate genes associated with resistant phenotypes can be used as positional and functional variation sites for further characterization of WWP major gene resistance against C. ribicola. Our results demonstrate that integration of RNA-seq-based transcriptome analysis and high-throughput genotyping is an effective approach for discovery of a large number of nucleotide variations and for identification of functional gene variants associated with adaptive traits in a non-model species.

Overexpression of a western white pine PR10 protein enhances cold tolerance in transgenic Arabidopsis

The PmPR10-1.10 protein from western white pine is known to be associated with frost hardiness, and up-regulated by seasonal cold acclimation and biotic and abiotic stresses. To gain insight into the molecular basis of cold hardiness, we investigated the potential physiological role of PmPR10-1.10 by gene overexpression in transgenic Arabidopsis plants. A binary vector was constructed for PmPR10-1.10 synthesis in higher plants and transgenic Arabidopsis lines were generated by Agrobacterium-mediated transformation. Following Western protein blot analysis confirming target protein production, transgenic Arabidopsis lines were tested for cold tolerance by electrolyte leakage analysis post treatment of different freezing temperatures. Our results demonstrate that accumulation of PmPR10-1.10 protein resulted in significantly greater freezing tolerance in transgenic plants than in wild type plants. This indicates that the transfer and selection of cold acclimation proteins like PmPR10-1.10 may be a breeding strategy for the development of freezing tolerance in conifers.