| Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst) is one of the mostdevastating diseases in wheat worldwide. Due to its fast spread and high frequent occurrences,the disease has attracted much global attention. However, the lacking of a stabletransformation system for Pst limited the systemic understanding of the mechanisms of itspathogenicity. Together with the rapid variation of Pst, control of the disease has been in apassive state. Therefore, it has become urgent to speed up the understanding of themechanisms of virulence variations of the pathogen.As an obligate biotrophic fungus, Pst completely depends on their living host tissue forgrowth and reproduction. During infection, Pst forms a specialized infection structure calledhaustoria to take nutrients and water from host plant tissue and establishes an intimate feedingrelationship. Cytological studies revealed that like other haustoria-forming pathogens, Pstreleases a series of secreted proteins from haustoria to host cells, but little is known about themechanisms by which the secreted proteins enter into and function inside plant cells to causethe stripe rust disease. Meanwhile, aside from the virulence role, when the host cellscontaining corresponding resistance (R) genes, effectors will be recognized by the hostresistance genes, which will trigger a hypersensitive response (HR). Thus, the effectors playan avirulence role. As a typical resistant reaction, how is the cell death initiated and how arethe signals transducted during HR are still unclear.Therefore, in this paper we intended to identify Pst effectors through large-scalescreening based on the available Pst genome data, analyze the virulence and avirulencefunctions of effectors and identify the motifs responsible for virulence and translocation.Moreover, we planned to identify genes which could induce host cell death from the cDNAlibraries of wheat-Pst interactions, analyze the cell death-induction mechanisms and theirroles in wheat-Pst interactions. This research might provide new insights in revealing thepathogenicity mechanisms of Pst and offer a new approach to engineer durable resistancethrough modulation of the pathogen genes, which is of great theoretical and practicalsignificance in the sustainable control of stripe rust.In this study, we have obtained the following results: 1. Bioinformatic analyses revealed723potential secreted proteins in the Pst genome, andfive were identified to be able to suppress the cell death caused by BAX in tobacco cells.qRT-PCR analyses showed that secreted protein genes Pst148and Pst9302were inducedduring the Pst infection with a peak at18hpi; Pst227was up-regulated during the entireinfection process; and Pst8853was induced and exhibited two peaks at the early and latestages of the infection, while Pst82was down-regulated during the infection. The differentexpression patterns of the effector genes studied during the infection process may representthe various functions of the effectors.Transient expression of the effectors in wheat via the bacteria Type III secretion system(TTSS) suppressed the host defense response triggered by Pst, with less H2O2accumulationand smaller cell death area. Transient expression of Pst8853triggered hypersensitive-like celldeath in the Yr27near isogenic line while no significant induction of cell death was observedfor the other four effectors expressed in43wheat cultivar lines carrying different resistantgenes, indicating a potential specific interaction between the gene products Pst8853in Pst andYr27in the wheat line. Transient expression of truncated versions of the effector genes intobacco revealed that the virulence motifs were located at the N-terminal11-20,1-23,21-40,21-40and21-30amino acids for Pst82, Pst148, Pst227, Pst9302and Pst8853, respectively,and no a common or conserved motif was observed.Subcellular localization of the effectors in tobacco cells showed that all of theeffectors-eGFP fusion proteins were located intracellular, indicating that the effectors weretranslocated into host cells independent of Pst. Mutation analysis revealed that thetranslocation motifs exist in the first40,17,60,30and40amino acids for secreted proteinsPst82, Pst148Pst227, Pst9302and Pst8853, respectively. Meanwhile, we verified that thepredicted translocation motif W/Y/FxC in the powdery mildew fungus, Blumaria graminis f.sp. tritici, was not essential for Pst effectors.2. Through transient expression in tobacco cells, two Pst genes, Pst322and PsANT, anda wheat gene, TaLls, were identified to be able to trigger cell death in tobacco and wheat.These genes encode a secreted protein with an unknown function, an adenine nucleotidetranslocase (ANT) and a lethal leaf-spot1, respectively. More detailed characteristics of thesegenes are the following:Pst322. qRT-PCR analysis showed that it was induced in-planta during the Pst infectionand reached a peak at18hpi, indicating that Pst322may involve in the early recognition ofPst by the host plant and in the regulation of the host defense response. Transient expressionof Pst322in wheat leaves via particle bombardment resulted in significant cell death in all25tested wheat cultivars. Moreover, the cell death induction function of Pst322was alsoobserved in non-host plants, including barley, tobacco and Arabidopsis thaliana. Thus, we inferred that Pst322function as an elicitor with a broad spectrum activity. Using the hostinduced gene silencing system (HIGS), Pst322was silenced during a compatible interaction.Histological observation showed that the silence of Pst322blocked the growth anddevelopment of Pst, with shorter initial invading hypha, more branches and smaller infectionarea, indicating that in addition to trigger the basal defense response in host, the gene alsoparticipates in the modulation of the differentiation and development of Pst.PsANT. The gene product contains three typical conserved repeat mito-carr proteindomains, which are conserved among animals, Caenorhabditis elegan and fungi.Immuno-cytochemical localization experiments showed the mitochondria localization ofPsANT in normal Pst invading hypha cells and cells undergoing collapse, implying its roles inenergy supply for the pathogen growth and cell death modulation for senescent cells.Transient expression of PsANT verified a pro-cell death role of PsANT in tobacco leaves aswell as in wheat leaves. Mutation analyses revealed a cumulative effect of the three conservedmotifs in cell death induction. qRT-PCR analyses showed that PsANT was induced at24hpiwith a peak at48hpi and kept expressed later on. Knockdown of PsANT during thecompatible wheat-Pst interaction by HIGS led to fewer branches and haustoria mother cells atthe early stage; shorter hyphae, more branches and haustoria at the middle stage; butrecovered Pst growth at the late stage, suggesting that PsANT may modulate the Pst growthvia supplying energy, but it may not be essential for the Pst pathogenicity.TaLls1. This gene encodes a pheophorbide a oxygenase (PaO). The qRT-PCRexperiments showed that TaLls1was up-regulated in the resistant wheat-Pst reaction, but nosignificant changes in the susceptible reaction. Transient expression of TaLls1in wheat andtobacco resulted in the accumulation of the red chlorophyll catabolite (downstream productsof PaO), which finally led to cell death. A mutant of the conserved Rieske [Fe-S] motif couldnot induce cell death, suggesting that Rieske [Fe-S] was essential for cell death induction.Silencing of TaLls1by virus-induced gene silencing (VIGS) in wheat led to the accumulationof pheophorbide a (upstream substrates of PaO) and finally caused leaf cell death withoutpathogen attacks. These results suggested a threshold for TaLls1in maintaining cellhomeostasis to adapt in various stresses, and shed light on the role of TaLls1in cell deathregulation. Furthermore, silencing of TaLls1in wheat enhanced tolerance to a virulent race ofPst, CYR31, via an significant increase in H2O2generation, elevated cell death, andup-regulation of pathogenesis-related genes, suggesting a negative role of TaLls1in wheatresistance to Pst. |
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