Identification And Functional Analysis Of Pathogenicity-related Genes Of Puccinia Striiformis F. Sp. Tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici(Pst), is an important fungal disease in wheat production. The utilization of disease-resistant varieties is the main method of controlling stripe rust in current wheat production. However, the resistance of wheat cultivar will be lost due to the constant pathogenicity variation of Pst, and so stripe rust continue to threaten global wheat production and global food security. The study for molecular mechanism of Pst pathogenicity and its variation is of great significance to explore the new strategies for durably controlling the disease.In this study, we did research in pathogenicity-related effector genes and Ras and kinase genes in cellular signal transduction, to analyze their functions in plant infection and pathogenicity of Pst. This study is great significance to explore the molecular mechanism of Pst pathogenicity, and also provide important theoretical basis for the development of new strategies for durably controlling the disease.1. Identification and functional analysis of effector genesWith the Agrobacterium-mediated PVX viral expression system, a total of 26 candidate Pst effector genes were transiently expressed in model plant-Nicotiana benthamiana. Six Pst effector genes were identificated due to they can suppress PCD triggered by BAX. All proteins(effector proteins) encoded by the 6 effector genes were proven to be secreted proteins by using the yeast secretory system. q RT-PCR analyses showed that 4 effector genes(Ps4884、Ps4941、Ps5278 and Ps5578) were induced in Pst inection of wheat, but Ps4593 and Ps5086 were down-regulated in Pst inection of wheat. Distinct expression profiles of Pst effector genes show suggest that these effector genes may function at different Pst infection stages.The virulence and avirulence functions of Pst effector genes were analyzed in host wheat plants by using the bacteria Type III secretion system(T3SS). The resluts showed that 5 effector genes could suppress wheat PTI triggered by non-pathogenic bacteria, and all 6 effector genes could suppress wheat ETI agianst an avirulent Pst isolate. These results prove that Pst effector genes have signicant virulence functions in wheat. On the other hand, large-scale screening showed that all Ps4884、Ps4941 and Ps5578 triggered HR-like cell death in the Yr1 near-isogenic line(NIL), and Ps4941 also triggered HR-like cell death in the Yr7 NIL. These reults indicate that Ps4884、Ps4941 and Ps5578 are candicate avirulence genes, and also suggest that different Pst avirulence genes may be recognized by a single rust resistance gene and that a single avirulence gene may be recognized by several rust resistance genes.Subcellular localization of the 6 Pst effector proteins in tobacco cells showed that all showed intracellular location in tobcco cells, and thus all of them are cytoplasmic effector proteins. A root uptake assay with GFP fusion proteins showed Pst effector proteins had the ability to carry the GFP-fusion proteins into root tip cells. These results indicate that Pst effector proteins can be translocated into plant cells in the absence of the pathogen. Sequence analysis shows that none of the 6 Pst effector proteins contains any putative RXLR motifs that are required for oomycete effector poteins. So it is likely that Pst and oomycete effector proteins show different translocation mechanism. There are 4 Pst effector proteins containing the Y/F/Wx C motif, which are reported in genomes of cereal powdery mildews. However, mutants in this motif could not change the subcellular localizations and ability to suppress the PCD triggered by BAX in tobacco of Pst effector proteins. Thus the Y/F/Wx C motif is not required for the translocation and virulence function of Pst effector proteins.2. Identification of wheat targets of key effector genesTwo key effector genes from the identified 6 Pst effector genes were selected for in-depth. Ps KE1(Ps5578) and Ps KE2(Ps4941) exhibited similar transcription patterns and virulence functions. They share low protein sequence identity but similar primary structure features. Knockdown of these two effectors by HIGS decreased the Pst disease in wheat. Histological observation showed that silenced plants also showed suppression of Pst hyphal growth and induction of several plant defenses. These data imply that Ps KE1 and Ps KE2 contribute to Pst virulence on wheat possibly by suppressing plant defense responses.Three wheat interaction proteins from different families were identified for Ps KE1 by uisng a yeast two-hybrid(Y2H) screen, including Ta PKDM7-1, Ta Trx-m4 and Ta DHN-1. Ta PKDM7-1 and Ta Trx-m4, but not Ta DHN-1, could also interact with Ps KE2. Further Y2 H assay showed that Ps KE1 did not interact with Ps KE2 and that none of the three host targets interacted with one another. Subcellular localization of the two effector proteins and the three wheat target proteins in wheat protoplast showed that both Ps KE1 and Ps KE2 localized to the cytoplasm and nucleus, and Ta PKDM7-1, Ta Trx-m4 and Ta DHN-1 also localized to cytoplasm, nucleus, and cytoplasm and nucleus, respectively. These data indicate that both effector proteins are capable of interacting with their host targets in the cytoplasm and/or the nucleus in vivo. Bimolecular fluorescence complementation(BIFC) assays confirm the interaction of the two effectors with their host targets in planta.q RT-PCR analyses showed all three wheat target genes were induced in wheat after Pst infection, and peaked time points coincided with the two effector genes. Knockdown of the three wheat target genes by virus induced gene silencing(VIGS) decreased the Pst disease in wheat. Histological observation showed that silenced plants also showed suppression of Pst hyphal growth and induction of several plant defenses. These data imply that the three targets contribute to wheat susceptibility to Pst, possibly by negatively regulating plant defense responses..3. Two distinct Ras genes exhibit differentiated functions in regulating pathogenicity and programmed cell deathThere are two distinct Ras genes deposited in the genome of Pst, Ps Ras1 and Ps Ras2. A phylogenetic analysis indicated that Ps Ras1 and Ps Ras2 fall in two distinct phylogenetic clades. Compared with Ras2 protein from other filamentous fungi, Ras2 protein from cereal rust fungi and the basidiomycete Ustilago maydis show unique deletions.q RT-PCR analyses showed that Ps Ras1 and Ps Ras2 had different expression profiles, and were induced and down-regulated in Pst inection of wheat, respectively. Knockdown of Ps Ras1 or Ps Ras2 by HIGS showed that the number of haustoria was significantly reduced in both Ps Ras1-silenced and Ps Ras2-silenced plants, and the decrease in hyphal length was clearly evident in Ps Ras2-silenced plants but not in Ps Ras1-silenced plants. Meanwhile, the disease phenotype was also significantly reduced in Ps Ras2-silenced plants but not in Ps Ras1-silenced plants. These results indicate that Ps Ras2 play a greater role in Pst pathogenicity than does Ps Ras1. However, Ps Ras2 could not complement the defect in the vegetative growth of the Fusarium graminearum ras2 mutant, supporting the functional difference in Ras2 between Pst and F. graminearum.Although Ps Ras1 plays a minor role in Pst pathogenicity, transient expression of Ps Ras1 but not Ps Ras2 in plants induced PCD, which was dependent on all of the conserved components of Ras GTPases in Ps Ras1 protein. The plant PCD triggered by Ps Ras1 showed similar morphological characteristics to the plant hypersensitive response(HR). In addition, it required the participation of plant MAPK cascades, which are also involved in HR, indicating that Ps Ras1-triggered PCD in plants is similar to HR.4. The cereal rust fungi-specific kinase gene Ps SRPKL is an important Pst pathogenicity factorA novel kinase gene Ps SRPKL, was identified from Pst haustoria-enriched transcripts. q RT-PCR analyses showed that Ps SRPKL was highly induced in Pst infection of wheat, and also induced in Pst infection of barberry, an alternate host of Pst. Ps SRPKL belongs to a group of protein kinase genes that are evolutionarily specific to cereal rust fungi, shown by a phylogenetic analysis. Further study showed that Ps SRPKL showed high levels of intra-species polymorphism and the mutant sequences were focused on its protein kinase domain. Subcellular localizations in tobacco and Arabidopsis showed that Ps SRPKL localizes to the nucleus. Meanwhile, overexpression of Ps SRPKL in the fission yeast induces aberrant morphology and decreases resistance to environmental stimuli(oxidative stress and hyperosmotic stress).Knockdown of Ps SRPKL by HIGS showed Ps SRPKL-silenced plants decreased the Pst disease in wheat. Histological observation showed that the silenced plants also compromised fungal growths and increased reactive oxygen species accumulation in host cells. Thus Ps SRPKL is proven to be an important Pst pathogenicity factor, which is responsible for fungal development and responses to environmental stresses, therefore contributing significantly to Pst pathogenicity on wheat.