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• W4288988319 abstract "Species of the genus Phytophthora, the plant killer, cause disease and reduce yields in many crop plants. Although many Resistance to Phytophthora infestans (Rpi) genes effective against potato late blight have been cloned, few have been cloned against other Phytophthora species. Most Rpi genes encode nucleotide-binding domain, leucine-rich repeat-containing (NLR) immune receptor proteins that recognize RXLR (Arg-X-Leu-Arg) effectors. However, whether NLR proteins can recognize RXLR effectors from multiple Phytophthora species has rarely been investigated. Here, we identified a new RXLR-WY effector AVRamr3 from P. infestans that is recognized by Rpi-amr3 from a wild Solanaceae species Solanum americanum. Rpi-amr3 associates with AVRamr3 in planta. AVRamr3 is broadly conserved in many different Phytophthora species, and the recognition of AVRamr3 homologs by Rpi-amr3 activates resistance against multiple Phytophthora pathogens, including the tobacco black shank disease and cacao black pod disease pathogens P. parasitica and P. palmivora. Rpi-amr3 is thus the first characterized resistance gene that acts against P. parasitica or P. palmivora. These findings suggest a novel path to redeploy known R genes against different important plant pathogens. Species of the genus Phytophthora, the plant killer, cause disease and reduce yields in many crop plants. Although many Resistance to Phytophthora infestans (Rpi) genes effective against potato late blight have been cloned, few have been cloned against other Phytophthora species. Most Rpi genes encode nucleotide-binding domain, leucine-rich repeat-containing (NLR) immune receptor proteins that recognize RXLR (Arg-X-Leu-Arg) effectors. However, whether NLR proteins can recognize RXLR effectors from multiple Phytophthora species has rarely been investigated. Here, we identified a new RXLR-WY effector AVRamr3 from P. infestans that is recognized by Rpi-amr3 from a wild Solanaceae species Solanum americanum. Rpi-amr3 associates with AVRamr3 in planta. AVRamr3 is broadly conserved in many different Phytophthora species, and the recognition of AVRamr3 homologs by Rpi-amr3 activates resistance against multiple Phytophthora pathogens, including the tobacco black shank disease and cacao black pod disease pathogens P. parasitica and P. palmivora. Rpi-amr3 is thus the first characterized resistance gene that acts against P. parasitica or P. palmivora. These findings suggest a novel path to redeploy known R genes against different important plant pathogens. IntroductionSpecies in the oomycete genus Phytophthora cause many devastating plant diseases. For example, P. infestans, P. parasitica, P. cactorum, P. ramorum, P. sojae, P. palmivora, and P. megakarya cause potato and tomato late blight, tobacco black shank disease, strawberry crown and leather rot, sudden oak death, soybean root and stem rot, and cacao black pod disease, respectively. P. infestans and P. sojae infect few plant species, while others such as P. parasitica, P. ramorum, and P. palmivora have a broad host range (Kamoun et al., 2015Kamoun S. Furzer O. Jones J.D.G. Judelson H.S. Ali G.S. Dalio R.J.D. Roy S.G. Schena L. Zambounis A. Panabières F. et al.The Top 10 oomycete pathogens in molecular plant pathology.Mol. Plant Pathol. 2015; 16: 413-434Crossref PubMed Scopus (451) Google Scholar).Plant immunity involves detection of pathogen-derived molecules by either cell-surface pattern recognition immune receptors or intracellular nucleotide-binding domain, leucine-rich repeat-containing (NLR) immune receptors, which activate either pattern-triggered immunity (PTI) or effector-triggered immunity (ETI), respectively (Jones and Dangl, 2006Jones J.D.G. Dangl J.L. The plant immune system.Nature. 2006; 444: 323-329Crossref PubMed Scopus (8005) Google Scholar). So far, more than 20 Resistance to P. infestans (Rpi) genes were cloned from wild Solanum species that confer resistance against potato late blight (Vleeshouwers et al., 2011Vleeshouwers V.G.A.A. Raffaele S. Vossen J.H. Champouret N. Oliva R. Segretin M.E. Rietman H. Cano L.M. Lokossou A. Kessel G. et al.Understanding and exploiting late blight resistance in the age of effectors.Annu. Rev. Phytopathol. 2011; 49: 507-531Crossref PubMed Scopus (276) Google Scholar). Several Resistance genes against P. sojae (Rps) have also been mapped in different soybean accessions, and a few were cloned (Sahoo et al., 2017Sahoo D.K. Abeysekara N.S. Cianzio S.R. Robertson A.E. Bhattacharyya M.K. A novel Phytophthora sojae resistance Rps12 gene mapped to a genomic region that contains several Rps genes.PLoS ONE. 2017; 12: e0169950Crossref PubMed Scopus (40) Google Scholar; Wang et al., 2021Wang W. Chen L. Fengler K. Bolar J. Llaca V. Wang X. Clark C.B. Fleury T.J. Myrvold J. Oneal D. et al.A giant NLR gene confers broad-spectrum resistance to Phytophthora sojae in soybean.Nat. Commun. 2021; 12: 6263-6268Crossref PubMed Scopus (8) Google Scholar). In tobacco, the black shank resistance genes Phl, Php, and Ph were genetically mapped, but not yet cloned; these confer race-specific resistance to P. parasitica (also known as [aka] P. nicotianae) isolates (Gallup and Shew, 2010Gallup C.A. Shew H.D. Occurrence of race 3 of Phytophthora nicotianae in North Carolina, the causal agent of black shank of tobacco.Plant Dis. 2010; 94: 557-562Crossref PubMed Scopus (23) Google Scholar; Bao et al., 2019Bao Y. Ding N. Qin Q. Wu X. Martinez N. Miller R. Zaitlin D. Li D. Yang S. Genetic mapping of the Ph gene conferring disease resistance to black shank in tobacco.Mol. Breeding. 2019; 39: 122https://doi.org/10.1007/s11032-019-1036-xCrossref Scopus (3) Google Scholar). For P. palmivora, some resistant cacao (Theobroma cacao) accessions were identified, but no dominant R genes have been defined or cloned (Thevenin et al., 2012Thevenin J.-M. Rossi V. Ducamp M. Doare F. Condina V. Lachenaud P. Numerous clones resistant to Phytophthora palmivora in the “Guiana” genetic group of Theobroma cacao L.PLoS One. 2012; 7: e40915Crossref PubMed Scopus (19) Google Scholar). In summary, apart from Rpi genes, very few R genes against Phytophthora pathogens have been cloned.Solanum americanum and Solanum nigrum are wild Solanaceae species and are highly resistant to P. infestans (Witek et al., 2016Witek K. Jupe F. Witek A.I. Baker D. Clark M.D. Jones J.D.G. Accelerated cloning of a potato late blight–resistance gene using RenSeq and SMRT sequencing.Nat. Biotechnol. 2016; 34: 656-660Crossref PubMed Scopus (138) Google Scholar, Witek et al., 2021Witek K. Lin X. Karki H.S. Jupe F. Witek A.I. Steuernagel B. Stam R. van Oosterhout C. Fairhead S. Heal R. et al.A complex resistance locus in Solanum americanum recognizes a conserved Phytophthora effector.Nat. Plants. 2021; 7: 198-208Crossref PubMed Scopus (14) Google Scholar). Two Rpi genes of coiled-coil (CC) type, Rpi-amr3 and Rpi-amr1, were cloned from different S. americanum accessions; both confer late blight resistance in cultivated potato (Witek et al., 2016Witek K. Jupe F. Witek A.I. Baker D. Clark M.D. Jones J.D.G. Accelerated cloning of a potato late blight–resistance gene using RenSeq and SMRT sequencing.Nat. Biotechnol. 2016; 34: 656-660Crossref PubMed Scopus (138) Google Scholar, Witek et al., 2021Witek K. Lin X. Karki H.S. Jupe F. Witek A.I. Steuernagel B. Stam R. van Oosterhout C. Fairhead S. Heal R. et al.A complex resistance locus in Solanum americanum recognizes a conserved Phytophthora effector.Nat. Plants. 2021; 7: 198-208Crossref PubMed Scopus (14) Google Scholar). S. nigrum is a hexaploid species that was thought to be a “non-host” plant of P. infestans. No Rpi gene had been cloned from S. nigrum until we reported the functional Rpi-amr1 homolog Rpi-nig1 (Witek et al., 2021Witek K. Lin X. Karki H.S. Jupe F. Witek A.I. Steuernagel B. Stam R. van Oosterhout C. Fairhead S. Heal R. et al.A complex resistance locus in Solanum americanum recognizes a conserved Phytophthora effector.Nat. Plants. 2021; 7: 198-208Crossref PubMed Scopus (14) Google Scholar).In oomycetes, the recognized effectors are usually secreted RXLR (Arg-X-Leu-Arg, X represents any amino acid)-EER (Glu-Glu-Arg) proteins that are translocated into plant cells (Rehmany et al., 2005Rehmany A.P. Gordon A. Rose L.E. Allen R.L. Armstrong M.R. Whisson S.C. Kamoun S. Tyler B.M. Birch P.R.J. Beynon J.L. Differential recognition of highly divergent downy mildew avirulence gene alleles by RPP1 resistance genes from two Arabidopsis lines.Plant Cell. 2005; 17: 1839-1850Crossref PubMed Scopus (319) Google Scholar; Wang et al., 2019Wang S. McLellan H. Bukharova T. He Q. Murphy F. Shi J. Sun S. van Weymers P. Ren Y. Thilliez G. et al.Phytophthora infestans RXLR effectors act in concert at diverse subcellular locations to enhance host colonization.J. Exp. Bot. 2019; 70: 343-356Crossref PubMed Scopus (34) Google Scholar). Dozens of Avirulence (Avr) genes encoding recognized effectors from Phytophthora species have been identified, and they are typically fast-evolving and lineage-specific molecules (Jiang et al., 2008Jiang R.H.Y. Tripathy S. Govers F. Tyler B.M. RXLR effector reservoir in two Phytophthora species is dominated by a single rapidly evolving superfamily with more than 700 members.Proc. Natl. Acad. Sci. USA. 2008; 105: 4874-4879Crossref PubMed Scopus (304) Google Scholar). Recently, AVRamr1 (PITG_07569), the recognized effector of Rpi-amr1, was identified by a cDNA pathogen enrichment sequencing approach (Lin et al., 2020Lin X. Song T. Fairhead S. Witek K. Jouet A. Jupe F. Witek A.I. Karki H.S. Vleeshouwers V.G.A.A. Hein I. et al.Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq).Mol. Plant Pathol. 2020; 21: 1502-1512Crossref PubMed Scopus (10) Google Scholar). Surprisingly, AVRamr1 homologs were identified from P. parasitica and P. cactorum genomes, and both are recognized by all Rpi-amr1 variants (Witek et al., 2021Witek K. Lin X. Karki H.S. Jupe F. Witek A.I. Steuernagel B. Stam R. van Oosterhout C. Fairhead S. Heal R. et al.A complex resistance locus in Solanum americanum recognizes a conserved Phytophthora effector.Nat. Plants. 2021; 7: 198-208Crossref PubMed Scopus (14) Google Scholar). Similarly, AVR3a-like effectors were found in different Phytophthora species, including P. capsici and P. sojae, and the recognition of AVR3a homologs correlates with P. capsici or P. sojae resistance in Nicotiana species and soybean (Shan et al., 2004Shan W. Cao M. Leung D. Tyler B.M. The Avr1b locus of Phytophthora sojae encodes an elicitor and a regulator required for avirulence on soybean plants carrying resistance gene Rps1b.Mol. Plant Microbe Interact. 2004; 17: 394-403Crossref PubMed Scopus (241) Google Scholar; Vega-Arreguín et al., 2014Vega-Arreguín J.C. Jalloh A. Bos J.I. Moffett P. Recognition of an Avr3a homologue plays a major role in mediating nonhost resistance to Phytophthora capsici in Nicotiana species.Mol. Plant Microbe Interact. 2014; 27: 770-780Crossref PubMed Scopus (48) Google Scholar). In addition, AVRblb2 homologs from P. andina and P. mirabilis trigger a hypersensitive response (HR) with Rpi-blb2 (Oliva et al., 2015Oliva R.F. Cano L.M. Raffaele S. Win J. Bozkurt T.O. Belhaj K. Oh S.-K. THINES M. Kamoun S. A recent expansion of the RXLR effector gene Avrblb2 is maintained in global populations of Phytophthora infestans indicating different contributions to virulence.Mol. Plant Microbe Interact. 2015; 28: 901-912Crossref PubMed Scopus (25) Google Scholar). Remarkably, a single N336Y mutation in R3a expands its recognition specificity to a P. capsici AVR3a homolog (Segretin et al., 2014Segretin M.E. Pais M. Franceschetti M. Chaparro-Garcia A. Bos J.I.B. Banfield M.J. Kamoun S. Single amino acid mutations in the potato immune receptor R3a expand response to Phytophthora effectors.Mol. Plant Microbe Interact. 2014; 27: 624-637Crossref PubMed Scopus (80) Google Scholar). These reports raise intriguing questions. Could RXLR effectors be widely conserved molecules among different Phytophthora species? Could these effectors be recognized by the same plant immune receptor? Of particular interest, could such effector recognition capacity enable disease resistance?Here we show that Rpi-amr3 confers resistance to all tested late blight isolates in both the field and laboratory conditions. We also identified AVRamr3, a novel AVR protein from P. infestans, by screening an RXLR effector library. AVRamr3 is a broadly conserved effector found in 13 different Phytophthora species. We also show functional Rpi-amr3 genes are widely distributed among S. americanum and S. nigrum accessions. The recognition of AVRamr3 not only enables resistance to a wide range of P. infestans isolates but also to other economically important Phytophthora pathogens such as tobacco black shank disease and cacao black pod disease pathogens P. parasitica and P. palmivora. Rpi-amr3 is the first reported R gene that confers resistance against P. parasitica and P. palmivora.ResultsRpi-amr3 confers late blight resistance in the laboratory and field conditionsRpi-amr3 was cloned by SMRT-RenSeq and reported to confer resistance against two Phytophthora infestans isolates, 88069 and 06_3928A, in a diploid potato line (Line 26, Solynta B.V.) (Witek et al., 2016Witek K. Jupe F. Witek A.I. Baker D. Clark M.D. Jones J.D.G. Accelerated cloning of a potato late blight–resistance gene using RenSeq and SMRT sequencing.Nat. Biotechnol. 2016; 34: 656-660Crossref PubMed Scopus (138) Google Scholar) in the laboratory conditions. However, whether Rpi-amr3 confers broad-spectrum and field resistance to late blight was not reported.To address this, we transformed Rpi-amr3 with its native promoter and terminator into a favored UK potato cultivar cv. Maris Piper. Two lines (SLJ24895-5C and SLJ24895-9A) (Supplemental Figure 1B) were selected for a field experiment in 2017, and SLJ24895-5C was further tested in the field in 2018 (Figure 1A ). These field trials indicate that Rpi-amr3 confers protection against potato late blight in field conditions, while the wild-type Maris Piper control lines were infected completely within ∼3 weeks once disease symptoms appeared (Figures 1A and 1B). As a result, the tuber yield of the Rpi-amr3 transgenic lines was significantly higher than the wild-type Maris Piper lines (Figures 1C and 1D). To determine the P. infestans genotypes present in the field trial, we sampled the infected leaves and genotyped them by SSR markers; most of the isolates corresponded to a dominant UK strain 6_A1 (aka Pink6) (Supplemental Table 1).To further evaluate the resistance spectrum of Rpi-amr3, we performed detached leaf assay (DLA) on SLJ24895-5C, with wild-type Maris Piper and Rpi-amr1 transgenic Maris Piper as controls. Seventeen P. infestans isolates with different origins and races were tested (Figure 1E, Supplemental Table 2). Our results show that Rpi-amr3 confers resistance against all tested isolates in potato in laboratory conditions but with different efficacy, and Rpi-amr3-mediated resistance is weaker than Rpi-amr1 in potato (Figure 1E). We also generated Rpi-amr3 stably transformed N. benthamiana lines. Two homozygous T2 lines, 13.3 and 16.5, were tested with nine P. infestans isolates. Both Rpi-amr3 transgenic N. benthamiana lines confer complete resistance to all tested P. infestans isolates (Supplemental Table 2).These data show that Rpi-amr3 confers potato late blight resistance in both the laboratory and field conditions.Avramr3 encodes a conserved RXLR-WY effector proteinTo identify the effector recognized by Rpi-amr3, we screened an RXLR effector library (Rietman, 2011Rietman H. Putting the Phytophthora Infestans Genome Sequence at Work: Multiple Novel Avirulence and Potato Resistance Gene Candidates Revealed.2011Google Scholar; Lin et al., 2020Lin X. Song T. Fairhead S. Witek K. Jouet A. Jupe F. Witek A.I. Karki H.S. Vleeshouwers V.G.A.A. Hein I. et al.Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq).Mol. Plant Pathol. 2020; 21: 1502-1512Crossref PubMed Scopus (10) Google Scholar) of 311 RXLR effectors by Agrobacterium tumefaciens-mediated co-expression with Rpi-amr3 in N. benthamiana. Most of these effectors (296/311) do not induce a HR when expressed alone or co-expressed with Rpi-amr3; 14 effectors are auto-active in N. benthamiana, and we found PITG_21190 specifically induces an HR with Rpi-amr3 (Figure 2A, Supplemental Table 3); therefore, we concluded that PITG_21190 is Avramr3. Avramr3 encodes a 339-amino acid (aa) protein with a signal peptide followed by RXLR, EER motifs, and four predicted WY motifs (Win et al., 2012Win J. Krasileva K.V. Kamoun S. Shirasu K. Staskawicz B.J. Banfield M.J. Sequence divergent RXLR effectors share a structural fold conserved across plant pathogenic oomycete Species.PLoS Pathog. 2012; 8: e1002400Crossref PubMed Scopus (106) Google Scholar) (Figure 2C). To characterize the expression profile of Avramr3, eight P. infestans isolates (T30-4, 88069, NL01096, 06_3928A, 6_A1, EC1, US23, and 99183) were used to inoculate a susceptible potato cultivar Maris Piper; RNA was isolated 2 days after the infection for RT–PCR. Our data show that Avramr3 are expressed in all eight isolates at 2 days after infection (Supplemental Figure 1A).Figure 2Identification and characterization of AVRamr3.Show full caption(A) Co-expression of Rpi-amr3:HA and AVRamr3:HIS-FLAG triggers cell death on N. benthamiana; Rpi-amr1 and AVRamr1 were used as controls. The photos were taken 3 days after infiltration; Agrobacterium strain GV3101(pMP90) carrying Rpi-amr3:HA or AVRamr3:HIS-FLAG constructs was used in this experiment. OD600 = 0.5. Three biological replicates were performed with the same results.(B) Co-expression of Rpi-amr3:HA and AVRamr3 truncations. All truncations are tagged with a C-terminal HIS-FLAG tag. T3, T4, T8, and T10 trigger cell death when co-expressed with Rpi-amr3, but not T1, T2, T5, T6, T7, and T9. Full-length AVRamr3:HIS-FLAG was used as control. OD600 = 0.5. Three biological replicates were performed with the same results.(C) Cartoon of AVRamr3 (PITG_21190), a protein with 339 aa with a signal peptide (lemon), RXLR-EER motif (green), and an effector domain (red) with four predicted WY motifs (details are shown in Supplemental Figure 4). T1–T10 indicates the AVRamr3 truncations used in HR assays. Those that induce HR after co-expression with Rpi-amr3 are marked by orange bars, and otherwise by blue.(D) Rpi-amr3::HA and AVRamr3::HIS-FLAG constructs were used for a bidirectional co-immunoprecipitation experiment, with Rpi-amr1-HA and AVRamr1::HIS-FLAG used as control. After HA pull-down of Rpi-amr3::HA or Rpi-amr1::HA, only AVRamr3::HIS-FLAG is associated with Rpi-amr3::HA. After Flag pull-down of AVRamr3::HIS-FLAG or AVRamr1-HIS-FLAG, only Rpi-amr3::HA is associated with AVRamr3::HIS-FLAG. Agrobacterium strain GV3101(pMP90) carrying different constructs was used for transient expression in the nrc2/3/4 knockout N. benthamiana line (210.4.3) to abolish the cell death phenotype. OD600 = 0.5. Three biological replicates were performed with the same results.(E) Rpi-amr3::Cluc and AVRamr3::Nluc constructs were used to test their interaction in planta; Rpi-amr1::Cluc and AVRamr1::Nluc were used as controls. The luciferase signal can be detected only on Rpi-amr3::Cluc and AVRamr3::Nluc co-expression. The nrc2/3/4 knockout N. benthamiana line (210.4.3) was used to abolish the cell death phenotype.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Many RXLR effectors are encoded by fast-evolving, multiple-member family genes with extensive sequence polymorphism, such as the Avr2 and Avrblb2 families (Gilroy et al., 2011Gilroy E.M. Breen S. Whisson S.C. Squires J. Hein I. Kaczmarek M. Turnbull D. Boevink P.C. Lokossou A. Cano L.M. et al.Presence/absence, differential expression and sequence polymorphisms between PiAVR2 and PiAVR2-like in Phytophthora infestans determine virulence on R2 plants.New Phytol. 2011; 191: 763-776Crossref PubMed Scopus (103) Google Scholar; Oliva et al., 2015Oliva R.F. Cano L.M. Raffaele S. Win J. Bozkurt T.O. Belhaj K. Oh S.-K. THINES M. Kamoun S. A recent expansion of the RXLR effector gene Avrblb2 is maintained in global populations of Phytophthora infestans indicating different contributions to virulence.Mol. Plant Microbe Interact. 2015; 28: 901-912Crossref PubMed Scopus (25) Google Scholar). To study the sequence polymorphism of Avramr3, we identified 17 additional Avramr3 homologs from 11 isolates from published databases (KR_1, 3928A, EC1, 6_A1, and US23) (Lee et al., 2020Lee Y. Cho K.-S. Seo J.-H. Sohn K.H. Prokchorchik M. Improved genome sequence and gene annotation resource for the potato late blight pathogen Phytophthora infestans.Mol. Plant Microbe Interact. 2020; 33: 1025-1028https://doi.org/10.1094/MPMI-02-20-0023-ACrossref PubMed Scopus (4) Google Scholar; Lin et al., 2020Lin X. Song T. Fairhead S. Witek K. Jouet A. Jupe F. Witek A.I. Karki H.S. Vleeshouwers V.G.A.A. Hein I. et al.Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq).Mol. Plant Pathol. 2020; 21: 1502-1512Crossref PubMed Scopus (10) Google Scholar) or cloned by PCR (EC1, NL01096, NL14538, 88069, PIC99183, and PIC99177) (Supplemental Figure 2). The sequence alignment shows Avramr3 is a highly conserved RXLR effector among P. infestans isolates, with only two polymorphic amino acids found among the 18 AVRamr3 homologs (Supplemental Figure 2).To define the domain responsible for recognition by Rpi-amr3, we fused 10 truncated Avramr3 fragments with HIS-FLAG tags and cloned into an expression vector with 35S promoter (T1–T10; Figure 2C; Supplemental Figure 3) and transiently co-expressed with Rpi-amr3 in N. benthamiana. Six AVRamr3 truncations (T1, T2, T5, T6, T7, and T9) cannot be recognized by Rpi-amr3 (Figure 2B). The protein levels of HIS-FLAG-tagged T6 and T7 are lower than others (Supplemental Figure 3A); therefore, we generated GFP-tagged constructs. The expression of T6-GFP is comparable with AVRamr3-GFP, but T7-GFP is not stable (Supplemental Figure 3B and 3C). We found four AVRamr3 truncations (T3, T4, T8, and T10) can be recognized by Rpi-amr3. T10 (111–240 aa), which carries the second and third WY motifs, is the minimal region to be recognized by Rpi-amr3, but not the adjacent T9 protein (130–258 aa) (Figure 2B). This suggests these 130 aa of AVRamr3 T10 are sufficient for recognition by Rpi-amr3 and initiation of HR.Rpi-amr3 is dependent on the helper NLRs NRC2, NRC3, and NRC4In Solanaceae, the functionality of many CC-NLR proteins requires helper NLR proteins of the NRC class (Wu et al., 2017Wu C.-H. Abd-El-Haliem A. Bozkurt T.O. Belhaj K. Terauchi R. Vossen J.H. Kamoun S. NLR network mediates immunity to diverse plant pathogens.Proc. Natl. Acad. Sci. USA. 2017; 114: 8113-8118Crossref PubMed Scopus (169) Google Scholar). To test whether Rpi-amr3 is NRC dependent, we co-expressed Rpi-amr3 and Avramr3 in NRC knockout N. benthamiana lines (nrc2/3_1.3.1, nrc4_185.9.1.3, nrc2/3/4_210.4.3) (Adachi et al., 2019Adachi H. Contreras M.P. Harant A. Wu C.-H. Derevnina L. Sakai T. Duggan C. Moratto E. Bozkurt T.O. Maqbool A. et al.An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species.Elife. 2019; 8: 121Crossref Scopus (69) Google Scholar; Wu et al., 2020Wu C.-H. Adachi H. De la Concepcion J.C. Castells-Graells R. Nekrasov V. Kamoun S. Kamoun S. NRC4 Gene cluster Is not essential for bacterial flagellin-triggered immunity.Plant Physiol. 2020; 182: 455-459Crossref PubMed Scopus (4) Google Scholar; Witek et al., 2021Witek K. Lin X. Karki H.S. Jupe F. Witek A.I. Steuernagel B. Stam R. van Oosterhout C. Fairhead S. Heal R. et al.A complex resistance locus in Solanum americanum recognizes a conserved Phytophthora effector.Nat. Plants. 2021; 7: 198-208Crossref PubMed Scopus (14) Google Scholar), as with wild-type N. benthamiana. We found HR on the nrc2/3_1.3.1 and nrc4_185.9.1.3 knockout lines, but not the nrc2/3/4_210.4.3 knockout lines. Similarly, only nrc2/3/4_210.4.3 knockout lines show susceptibility to P. infestans after Rpi-amr3 transient expression (Supplemental Figure 4). These data suggest both Rpi-amr3-mediated effector recognition and resistance are supported by either NRC2, NRC3, or NRC4.Rpi-amr3 associates with AVRamr3 in plantaTo date, most Rpi proteins recognize their cognate effectors in an indirect manner, apart from the RB and IPI-O effectors (Chen et al., 2012Chen Y. Liu Z. Halterman D.A. Molecular determinants of resistance activation and suppression by Phytophthora infestans effector IPI-O.PLoS Pathog. 2012; 8: e1002595Crossref PubMed Scopus (71) Google Scholar; Zhao and Song, 2021Zhao J. Song J. NLR immune receptor RB is differentially targeted by two homologous but functionally distinct effector proteins.Plant Commun. 2021; 2: 100236Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). To test the interaction between Rpi-amr3 and AVRamr3, we generated and transiently co-expressed Rpi-amr3:HA and AVRamr3:HIS-FLAG epitope-tagged constructs in nrc2/3/4 knockout N. benthamiana leaves to avoid cell death. Protein was then extracted, and bi-directional co-immunoprecipitations (Co-IPs) were performed. These Co-IPs indicate that Rpi-amr3 associates with AVRamr3 bidirectionally (Figure 2D). We also tested their interaction using a split-luciferase assay. Rpi-amr3:C-luciferase (Cluc) and AVRamr3:N-luciferase (Nluc) constructs were generated and transiently expressed in the nrc2/3/4 knockout N. benthamiana. Luciferase signal was detected only when Rpi-amr3:Cluc and AVRamr3:Nluc were co-expressed (Figure 2E), but not in the negative controls. These data suggest Rpi-amr3 associates with AVRamr3 in planta, but do not exclude the possible involvement of additional proteins.Avramr3 orthologs occur in multiple Phytophthora speciesTo study the evolution of Avramr3 in Phytophthora species, we searched for Avramr3 homologs from published Phytophthora and Hyaloperonospora arabidopsidis genomes. Surprisingly, we found Avramr3 homologs in many Phytophthora genomes, including P. parasitica, P. cactorum, P. palmivora, P. pluvialis, P. megakarya, P. litchii, P. ramorum, P. lateralis, P. sojae, P. capsici, and P. cinnamomi and in H. arabidopsidis. Most of the Avramr3 homologs are located at a syntenic locus (Figure 3A ). Notably, the P. infestans Avramr3-containing contig was not fully assembled; it lacks sequences on the 5′ side of Avramr3 (Figure 3A).Figure 3AVRamr3 is a conserved effector among different Phytophthora species.Show full caption(A) The synteny map of Avramr3 loci from 12 different Phytophthora genomes. The Avramr3 loci were extracted from different genomes, annotated by the gene prediction tool in EumicrobeDB, and then analyzed and visualized by Clinker. Avramr3 homologs are shown by purple triangles and indicated by a black arrow; the flanking genes with homology are represented by the corresponding colors. The Phytophthora clades are adapted from the Phytophthora database (Rahman et al., 2014Rahman M.Z. Uematsu S. Takeuchi T. Shirai K. Ishiguro Y. Suga H. Kageyama K. Two new species, Phytophthora nagaii sp. nov. and P. fragariaefolia sp. nov., causing serious diseases on rose and strawberry plants, respectively, in Japan.J. Gen. Plant Pathol. 2014; 80: 348-365Crossref Scopus (13) Google Scholar).(B) Expression of AVRamr3 homologs with HIS-FLAG tag alone does not trigger cell death on Nicotiana benthamiana. Agrobacterium strain GV3101(pMP90) carrying different constructs was used in this experiment. OD600 = 0.5. Three biological replicates were performed with the same results.(C) Co-expression of HIS-FLAG-tagged AVRamr3 homologs with Rpi-amr3::GFP in N. benthamiana. The AVRamr3 homologs from P. infestans (Pi), P. parasitica (Pp), P. cactorum (Pc), P. palmivora (Ppal), P. megakarya (Pmeg), P. litchii (Plit), P. sojae (Ps), P. lateralis (Plat), and P. pluvialis (Pplu) induce cell death after co-expression with Rpi-amr3::GFP, but not AVRamr3 homologs from P. ramorum (Pr), P. capsici (Pcap), and H. arabidopsidis (Hpa). The AVRamr3 homolog from P. cinnamomi (Pcin) shows an intermediate cell death. Agrobacterium strain GV3101(pMP90) carrying different constructs was used in this experiment. OD600 = 0.5. Three biological replicates were performed with the same results. The protein expression of the AVRamr3 homologs with HIS-FLAG tag was shown in Supplemental Figure 6.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To test whether those AVRamr3 homologs from different Phytophthora species are also recognized by Rpi-amr3, we synthesized and cloned them into an expression vector with the 35S promoter and performed transient expression assays in N. benthamiana. Expressing the effectors alone does not trigger HR in N. benthamiana (Figure 3B), but AVRamr3 homologs from P. parasitica, P. cactorum, P. palmivora, P. megakarya, P. litchii, P. sojae, P. lateralis, and P. pluvialis can induce HR when co-expressed with Rpi-amr3, respectively. The AVRamr3 homolog from P. cinnamomi triggers a weaker HR compared with other recognized AVRamr3 homologs, and the AVRamr3 homologs from P. ramorum, P. capsici, and H. arabidopsidis (Figure 3C) do not trigger Rpi-amr3-dependent HR. All these AVRamr3 homologs carry multiple WY motifs, but many polymorphic amino acids are present among these homologs. We" @default.
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• W4288988319 title "A potato late blight resistance gene protects against multiple Phytophthora species by recognizing a broadly conserved RXLR-WY effector" @default.
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• W4288988319 doi "https://doi.org/10.1016/j.molp.2022.07.012" @default.
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