Screening For Genes Involved In Resistance Against Leaf Rust In Populus Tomentosa Carr. And Characterization Of NBS Type Of Disease Resistance Genes

Populus tomentosa Carr. is an indigenous tree species of white poplar (in section Leuce) in China and widely employed for forestation and landscape in northern China. However, this poplar species is susceptible to a wide variety of diseases, particularly the leaf rust caused by Melampsora magnusiana Wagner, which often severely affect the poplar growth and yield. In contrast to tremendous progress in the identification and characterization of resistance loci in poplars (in section Tacamahaca and Aigeiros) as well as of putative disease resistance genes from genome sequences of P. trichocarpa, little is known about disease resistance in P. tomentosa so far.In this study, inoculation bioassays were performed on hybrids of P. tomentosa with leaf rust fungi and screened out 2 highly resistant clones of white poplars. Many resistance gene analogs (RGAs) were isolated from one resistant clone using PCR-based approach for the analyses of evolution and expression profile of disease resistance genes in white poplar and one RGA (DQ324288) was identified that showed putative involvement in resistance against leaf rust in P. tomentosa. Two members of DQ324288 gene family were cloned using RACE-PCR analysis and the expression profile of these 2 genes was analyzed using real time PCR analyses. Based on the construction of prokaryotic expression vector, RNA interference vector and sense expression vector, prokaryotic expression analysis and genetic transformation on resistant poplar and tobaccos were conducted to reveal the potential function of the above two genes. In addition, a number of resistance genes were identified from the genome of P. trichocarpa based on the RGAs of white poplar, and organization, evolution as well as expression profile of these genes in a triploid white poplar clone were studied. The main results from the above mentioned studies are described as below:1. The inoculation assays with leaf rust fungi were performed on 28 clones of hybrids of P. tomentosa and identified 13 susceptible clones with various degrees of rusting sypmton and 15 resistant clones without sympton, of which 2 clones of triploid white poplars [(P. tomentosa × P. bolleana) × p tomentosa] were considered highly resistant to leaf rust since hypersensitive response was observed. Based on the highly resistant poplars and presence of a nucleotide binding site (NBS) domain in majority of cloned plant disease resistance genes (R genes), 59 resistance gene analogs (RGAs) were identified by using PCR analysis with degenerate primers. The 59 RGAs were phylogenetically classified into 10 subfamilies and 54 RGAs with open reading frames (ORFs) were further grouped into two classes, toll and interleukin-1 receptor (TIR) and non-TIR. BLAST searches with reference to the genomic sequence of P. trichocarpa found 96 highly homologous regions distributed in 37 loci, suggesting the abundance and divergence of NBS encoding genes in the triploid poplar genome. Within subfamilies 1 to 3, the average nonsynonymous/synonymous substitution (ω) rates were < 1, indicating purifying selection on these RGAs, but some sites were clearly under diversifying selection with ω > 1. Many intergenic exchanges were also detected among these RGAs, indicating the probable role in homogenizing the NBS domains. Quantitative real-time PCR analysis revealed dramatic variations in the transcript level of 18 RGAs in the mature leaves, bark and roots of the triploid poplar and identified 2 RGAs that had significantly higher level of transcripts in bark, 4 RGAs in mature leaves, and 14 in the above ground portion of poplars, suggesting their probable roles respectively involved in resistance against the pathogens attacking the organs.2. A RGA (DQ324288 gene) was identified out of triploid poplar RGAs that shared high similarity (93%) with the genomic sequence of P. deltoides cultivar S9-2 MER locus, conferring resistance to three races of rust fungi M. larici-populina Kleb, and with the 60I2G11 gene within the MER locus. DQ324288 had homologous genes in twenty-eight triploid poplar clones, and was expressed constitutively and specifically in leaf tissue. Its expression level was increasing along with the increase in the level of resistance of the hosts against leaf rust, indicating its close relationship with the resistance against this pathogen. RACE-PCR analysis revealed two members of this gene family: PtDRGOl and PtDRG02 gene, encoding TIR-NBS-LRR and TIR-NBS proteins, respectively. These two genes displayed similar expression profile with DQ324288 gene, and positive responses to wounding, MeJA and SA rather than darkness and A. tumefaciens. Bioinformatic analysis showed that the deduced proteins of two genes were soluble and hydrophilic with respective pHi 8.165 and 10.325. The high level expression of fusion proteins of two genes in Escherichia coli was induced the IPTG and the expression level was elevated with the increase in induction temperature and time. The PtDRGOl gene was cloned into an expression vector and transferred to tobaccos via A. tumefaciens-mediated transformation. The integration of foreign genes into the genome of transgenic tobaccos was confirmed by PCR analyses with two sets of primers and the number of foreign genes in the genome of tobaccos were identified to be0.1-fold or 0.2-fold of that of native ACTIN gene by quantitative real-time PCR analysis. The expression of PtDRG01 gene varied dramatically among different transgenic lines. One-week-period inoculation with tobacco mosaic virus (TMV) revealed that the transgenic tobaccos contained less number of viruses than non-transgenic ones and that the number of viruses was negatively correlated with the expression level of PtDRG01 gene. Moreover, six-week-period inoculation induced abnormal morphology of apical leaves on the non-transgenic tobaccos and normal morphology of apical leaves on the transgenic tobaccos with high level transcripts of PtDRG01 gene, indicating that PtDRG01 gene has the potential to enhance resistance of tobaccos to TMV. In addition, a RNA interference expression vector of PtDRG gene family was constructed and transferred into the resistant triploid poplar through particle bombardment. Many transgenic poplars were obtained and the integration of RNAi sequences into the host genome was confirmed by the PCR analysis with two sets of gene specific primers.3. A total of 74 R genes with the NBS domains were identified from the genomic sequences of P. trichocarpa that were highly homologous to the RGAs from triploid white poplar. The extrons and introns in these genes varied significantly in both length and number, and the protein domains showed dramatic variations in organization, number and length. These 74 R genes were structurally classified into 9 classes with distinct protein domain organizations and phylogeneitcally divided into 11 subfamilies according to the degree of the nucleotide sequence similarity. Within 8 groups of genes with high similarity in nucleotide sequences and length, the average ω rates were > 1, < 1, or close to 1, respectively, indicating the positive selection, purifying selection or neutral selection on these resistance genes, but many sites within genes of 7 groups (except for group 6) were clearly under diversifying selection with co > 1. Many intergenic exchanges were also detected among these resistance genes, indicating the important roles that gene conversions play in the evolutionary process of resistance genes from P. trichocarpa. For a better understanding of resistance genes in white poplars, the expression patterns of these 74 genes in various organs of a triploid white poplar, under different growth conditions, were examined by using quantitative real-time PCR. Twenty-seven of 74 genes from P. trichocarpa were expressed in 6 examined organs at various levels. Six, two and three genes, respectively, displayed significantly higher expression levels in apical leaves, young bark and mature bark than in other organs, indicating that these genes may be involved in organ specific disease resistance. Twenty-four genes had dramatically greater expression in apical leaves than in mature leaves, 22 genes higher in the mature bark than in the young bark, and five genes systematically displayed dramatically stronger expression than other genes. Wounding induced an increase in the transcript level of 22 genes and a reduction for 2 genes. Twenty genes were up-regulated by darkness, 14 by methyl jasmonate acid (MeJA), 6 by salicylic acid (SA), and 11 by the compatible Agrobacterium tumefaciens, implying a complex interconnecting signal transduction pathways that regulates the expression of poplar R genes.Our results shed light on the genetic resources of poplar resistance, the evolution and expression profiling of poplar resistance genes, and the preliminary features of putative resistance gene against leaf rust in P. tomentosa, which will be helpful for the further characterization of their function.