The ESTs And Defense-related Genes Of Host In The Interaction Between Poplar And Melampsora Larici-populina

Poplar leaf rust, caused by Melampsora larici-populina(MLP), is a devastating disease leading severe economic loss in poplar plantation worldwide, with the characteristics of long distance transmission. Breeding the resistance cultivars is considered to be the most economical, effective, friendly and sustainable way to control and decrease the poplar rust disease. However, M. larici-populina behaves the features of a heteroecious life cycle, fast variation and continue development of new virulent race, which are slowed down and limited the selection process of poplar resistance varieties. Therefore, deciphering the expression profiles of poplar-rust interactions, especially on cloning and characterizing the defense-related(DR) genes, as well as analyzing functions of these genes, could provide basic information for understanding the host defense responsive and resistance mechanism in the poplar-rust interaction, which also provide basic rationale and technical support for selecting resistant cultivars. In this study, Populus szechuanica leaves infected by avirulent isolate Sb052 of M. larici-populina was used as the materials. An incompatible P. szechuanica-MLP interaction expressed sequence tag(ESTs) library was constructed by suppression subtraction hybridization(SSH). Rapid amplification of c DNA ends(RACE) was performed to clone P. szechuanica DR(Ps DR) genes, and with the bioinformatics methods were used to identify characterization and functions of these genes. In addition, the spatio-temporal expression patterns of these defense-related genes were analysed in the interaction of P. szechuanica and MLP. The main results were as followed:1. An incompatible interaction SSH-c DNA library was conducted using the mixture P. szechuanica leaves inoculation with isolate Sb052 at 0.5、1、2、3 and 4 dpi. 515 clones in the library were sequenced, and141 unigenes including 66 contigs and 75 singletons were acquired after cluster analysis. Results of function annotation and category showed that in those function of known unigens, the most abundant(30%) category was realated with the metabolic process, 24% were involved in cellular process, 10% were responsed to stimulus, and 19% unigenes were involved in the signal transformation, localization, immune system process, biological regulation, developmental and death process.The analysis of resistance related genes in our library showed that the most abundant ESTs(with 41 copies) encoded the 1-aminocyclopropane-1-carboxylate oxidase. The NBS-type disease resistance genes contained 18 copies, β-1,3-glucanase(GNS) and phenylalanine ammonnia lyase(PAL) contained 13 and 10 copies, respectively. In addition, other DR genes were also found in the library such as pathogenesis-related protein 1(PR1), thaumatin-like protein(TLP),ABC transporter genes. We supposed these genes were involved the P. szechuanica defense response and played important role during the resistance and defense response to the rust fungi.2. Based on the ESTs sequences of P. szechuanica pathogen-related protein 1(Ps PR1), β-1,3-glucanase(Ps Gns), thaumatin-like protein(Ps TLP) and phenylalanine ammonia-lyase(Ps PAL) gens in our library, RACE was used to clone these genes. The full-length c DNA sequences of Ps PR1, Ps Gns, Ps TLP1, Ps TLP2 and Ps PAL genes were 728, 1189, 929, 885 and 2586 bp, respectively. Predicted proteins encoded by Ps PR1, Ps Gns and Ps TLPs genes were contained a signal putative signal peptide, respectively, and conserved in the Populus species. The predict proteins encoded by Ps PR1, Ps Gns, Ps TLPs and Ps PAL genes were belonged to SCP, Glyco-hydro-17, GH64-TLP-SF and Lyase_I_like superfamily, respectively, and SCP, GH64-TLP-SF and Lyase_I_like superfamily contained functional motifs such as CAP, TLP-PA and THN, and PAL-HAL, respectively.3. RT-q PCR was performed to analysis the spatio-temporal expression patterns of Ps PR1, Ps Gns, Ps TLP1, Ps TLP2, Ps PAL, nonexpressor of pathogenesis_relatedgenes1(NPR1) and ABC transpoter genes in the P. szechuanica-MLP interaction. As the results showed that these genes expression induced by MLP were increased at 0.5 dpi, and increased levels in incompatible interaction were normally higher than in compatible interaction. In the incompatible interaction, the expression level of most Ps DR genes was reached two peaks at 0.5 and 2 dpi or 4 and 7 dpi respectively; the expression levels of Ps PR1, Ps TLP2, Ps PAL, NPR1 and ABC transporter genes were increased most in the petioles, followed by in the top buds and the fewest or even not detected in the roots.4. Four P. szechuanica peroxiredoxin(Ps Prx) genes were cloned. The full-length c DNAs of Ps1 Cys Prx, Ps2 Cys Prx, Ps Prx Q and Ps Prx II were 930,1150, 975 and 853 bp, respectively. The predict proteins encoded by 4 Ps Prx genes were belonged to thioredoxin superfamily, and contained different functional site such as peroxidatic cysteine or resolving cysteine. Different type of Ps Prx contained different conserved motifs such as PVCTTE、PXXXTXXC and CXXC, and we supposed that Ps Prx genes could be involved in redox signaling pathways of P. szechuanica to defense the fungi pathogen.5. After induced by the avirulent isolate Sb052 of MLP, Ps Prx genes were the highest expression in the leaves, followed by the leaves next to the inoculation leaves, roots and top buds. In P. szechuanica leaves of the incompatible interaction, the relative expression levels of Ps Prx genes were obviously up-regulated during 1~7 dpi, which had two peaks and increased higher than in the compatible interaction.