The Translocation Machinery Analysis Of Oomycete And Fungal Effectors

Fungi and oomycete are phylogeneticlly distinct eukaryotic microorganisms, includingmany plant pathogens that cause destructive diseases and tremendous economic losses to theagricultural production. In crop production, one of most effective approaches to control plantdiseases is to take advantage of resistant varieties. The gene-for-gene relationship betweenhost plants and pathogens is extensively described in many plant pathosystems. Resistanceproteins (R) expressed in resistant varieties are capable of recognizing the correspondingavirulence proteins (Avr) encoded by pathogens that is required to initiate hypersensitiveresponse (HR) and prevent infection. The accelerated characterization of R-Avr gene pairshas broadened our view about recognition mechanism between plants and pathogens. Withrapid development in studying Avr genes of plant pathogens, it has become evident that Avrgene coding products also contribute to pathogen virulence on plants lacking cognate Rproteins. As a result, the Avr gene was re-named as effector gene, which is neutral term anddo not imply negative and positive impact on the outcome of the pathogen-host interaction.Large-scale genome sequencing has led to the prediction of an arsenal of effector genes indiverse plant pathogens, among which the cloned Avr genes are just several special cases.Deciphering the precise bioactivities of effector proteins to understand how theyinterfere the plant defense responses and facilitate successful proliferation became a centralfocus in the past two decades. Since the majority of plant resistance proteins againstoomycete and fungal pathogens belong to nucleotide binding site leucine-rich repeat (NBS-LRR) proteins that are predicted to be located in the cytoplasm, the recognition of Avrproteins and R proteins occurs inside plant cells, suggesting that pathogen effector proteinsare translocated into host plant cells. Therefore, elucidation of the translocation mechanismof oomycete and fungal effector proteins and their roles in disease development will offernovel opportunities to understand how the pathogens manipulate host cells to establish aparasitic relationship and how to develop durable disease control measures. In the first part,we analyzed the expression profile and virulence activities of three predicted Phytophthorasojae cytoplasmic effectors after being delivered into plant cells via conserved RXLR motif during infection. In the second part, we concentrated on identification of the effectortranslocation machinery of plant pathogenic fungi. We further discovered that the fungal andoomycete pathogens shared conserved lipid raft-mediated endocytosis for effectortranslocation. The main results and conclusion are as follows:1. Three most up-regulated P. sojae Avh (Avirulence homologues) genes encoding RXLReffectors of during infection derived from microarray data were selected for functionalcharacterization. RT-PCR analysis showed that Avh238and Avh240were up-regulated during6-12h post-inoculation and Avh181up-regulated during12-24h post-inoculation. Theexpression levels of all three genes dropped from24h after inoculation. All three genes wereshown to be unable to suppress BAX triggered cell death whereas Avh238and Avh181of theP. sojae strain P6497induced cell death on soybean leaves. The predicted Avh238andAvh181proteins contain several sequence polymorphisms at their C-termini, which indicatedthat these two effectors were under positive selection. The Avh181(strain P7074, GenotypeII) protein with sequence variations was still able to induce cell death on soybean leaves,whereas Avh238(P7074, genotype II) with sequence variations was unable to induce celldeath.2. Several substitutive residues within the RXLR motif were defined to permit cell entry. Theresults revealed that lysine or histidine could replace the arginine at position1in the motif,that any large hydrophobic residue (isoleucine, methionine, phenylalanine, or tyrosine) couldreplace the leucine at position3. At position4, all residues tested allowed function. This ledto identification and functional validation of RXLR variants in fungal effectors for proteintranslocation. AvrPita protein from Magnaporthe oryzae contains KRRAYIER, RAALK andRLFKLIFK motif. AvrP123and AvrP4proteins from Melampsora lini contain RLTQ andRDIQ respectively. AvrM of M. lini contains KDMK, KALK, KLGT, RGFLR, KMKFLKand KLST motifs. Both bombardment cell re-entry assay and recombined protein root uptakeassay demonstrated that these RXLR variants are capable of delivering reporter protein intoplant cells. The RXLR similar motifs of AvrL567enable binding to phosphatidylinositol-3-phosphate (PI3P) and being transferred into host cells via lipid raft mediated endocytosis.These results suggest that PI3P mediated entry may be widespread in plant pathogenesis. Inaddition, some fungal effectors such AvrPita, AvrP123, AvrP4and AvrM showed high affinityto phosphatidic acid (PA) suggesting that PA is likely involved in the internalization ofeffectors into host plant cells or PA is cytoplasmic target of effectors.3. The Ps87gene of wheat stripe rust pathogen Puccinia striiformis f.sp. tritici encodes aprotein that is conserved in diverse fungal pathogens. Ps87homologs from a clade containingrust fungi are predicted to be secreted. Yeast signal sequence trap assay confirmed that the rust protein Ps87could be secreted from yeast cells, but a homolog from Magnaporthe oryzaethat was predicted to be nonsecreted, could not. Bombardment mediated cell re-entry assayand protein uptake assay revealed that a region of Ps87containing a conserved RXLR-likemotif [K/R]RLTG was capable of delivering oomycete effector Avr1b into soybean leaf cellsand carrying GFP into soybean root cells. The GFP fused N-terminus of Ps87specificallybinds to phosphatidic acid (PA) and phosphatidylinositol-3-phosphate (PI3P). Mutations inKRLTG motif abolish the ability to transport the reporter into plant cells and binding tophopholipids. The results suggest that Ps87and its secreted homologs could utilize similarprotein translocation machinery as those of oomycete and other fungal pathogens. However,Ps87did not show direct suppression activity on plant defense responses induced by both thepathogen associated molecular patterns (PAMPs) and the effectors. Transient expression ofPs87in tobacco leaves did not affect the expression of pathogenesis related (PR) genes andthe infection process of Phytophthora parasitica. These results suggest Ps87may representan "emerging effector" that has recently acquired ability to enter plant cells but has not yetacquired ability to alter host cell physiology.