Received: 11 July 2020 | Revised: 7 November 2020 | Accepted: 23 November 2020 DOI: 10.1111/mpp.13030 ORIGINAL ARTICLE Identification of RipAZ1 as an avirulence determinant of Ralstonia solanacearum in Solanum americanum Hayoung Moon1 | Ankita Pandey1 | Hayeon Yoon1 | Sera Choi1 | Hyelim Jeon2,3 | Maxim Prokchorchik1 | Gayoung Jung1 | Kamil Witek4 | Marc Valls 5,6 | Honour C. McCann7,8 | Min-Sung Kim1,9 | Jonathan D. G. Jones4 | Cécile Segonzac2,3,10 | Kee Hoon Sohn 1,11 1 Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea 2 Department of Agriculture, Forestry and Bioresources, Seoul National University, Seoul, Republic of Korea 3 Plant Immunity Research Center, Seoul National University, Seoul, Republic of Korea 4 The Sainsbury Laboratory, University of East Anglia, Norwich, UK 5 Department of Genetics, University of Barcelona, Barcelona, Spain 6 Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra, Spain 7 New Zealand Institute of Advanced Studies, Massey University, Auckland, New Zealand 8 Max Planck Institute for Developmental Biology, Tübingen, Germany 9 Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Republic of Korea 10 Department of Plant Science, Plant Genomics and Breeding Institute, Agricultural Life Science Research Institute, Seoul National University, Seoul, Republic of Korea 11 School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Republic of Korea Correspondence Kee Hoon Sohn, Department of Life Abstract Sciences, Pohang University of Science and Ralstonia solanacearum causes bacterial wilt disease in many plant species. Type Technology, Pohang 37673, Republic of Korea. III-secreted effectors (T3Es) play crucial roles in bacterial pathogenesis. However, Email: khsohn@postech.ac.kr some T3Es are recognized by corresponding disease resistance proteins and activate Funding information plant immunity. In this study, we identified the R. solanacearum T3E protein RipAZ1 National Research Foundation of Korea; (Ralstonia injected protein AZ1) as an avirulence determinant in the black nightshade Rural Development Administration, Grant/ Award Number: PJ01317501; National species Solanum americanum. Based on the S. americanum accession-specific aviru- Research Foundation, Grant/Award lence phenotype of R. solanacearum strain Pe_26, 12 candidate avirulence T3Es were Number: PID2019-108595RB-I00, 2015 and 0533; Seoul National University; Generalitat selected for further analysis. Among these candidates, only RipAZ1 induced a cell de Catalunya death response when transiently expressed in a bacterial wilt-resistant S. americanum accession. Furthermore, loss of ripAZ1 in the avirulent R. solanacearum strain Pe_26 resulted in acquired virulence. Our analysis of the natural sequence and functional variation of RipAZ1 demonstrated that the naturally occurring C-terminal truncation results in loss of RipAZ1-triggered cell death. We also show that the 213 amino acid central region of RipAZ1 is sufficient to induce cell death in S. americanum. Finally, we show that RipAZ1 may activate defence in host cell cytoplasm. Taken together, our data indicate that the nucleocytoplasmic T3E RipAZ1 confers R. solanacearum This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd Mol Plant Pathol. 2021;22:317–333.  wileyonlinelibrary.com/journal/mpp | 317 318 | MOON et al. avirulence in S. americanum. Few avirulence genes are known in vascular bacterial phytopathogens and ripAZ1 is the first one in R. solanacearum that is recognized in black nightshades. This work thus opens the way for the identification of disease resistance genes responsible for the specific recognition of RipAZ1, which can be a source of resistance against the devastating bacterial wilt disease. KEYWORDS avirulence, bacterial wilt, effector, effector-triggered immunity, Ralstonia solanacearum, Solanum americanum 1 | I NTRO D U C TI O N in plants. For example, Pseudomonas outer protein P2 (PopP2, also known as RipP2) is an acetyltransferase that is recognized by the Many bacterial pathogens translocate a suite of virulence proteins paired NLR proteins RPS4 (resistance to Pseudomonas syringae 4) and termed effectors into invading host cells via a type III secretion sys- RRS1 (resistance to Ralstonia solanacearum 1) in Arabidopsis thaliana tem to suppress plant defence responses such as the generation of (Deslandes et al., 2003, 2003; Narusaka et al., 2009). On translo- reactive oxygen species (ROS) (Jwa & Hwang, 2017; Macho & Zipfel, cation in the plant cell nucleus, PopP2 acetylates the RRS1 WRKY 2015). However, some effectors are recognized by corresponding DNA-binding domain, reconfigures RRS1, and activates RPS4- plant disease resistance (R) proteins that activate effector-triggered dependent immunity (Guo et al., 2020; Le Roux et al., 2015; Sarris immunity (ETI), resulting in restriction of pathogen proliferation et al., 2015). Another R. solanacearum effector, Ralstonia injected (Cesari, 2018; Jones et al., 2016). The effectors that are recognized protein B (RipB), triggers Roq1 (Recognition of XopQ 1)-dependent by R proteins are termed avirulence (Avr) proteins. The intracellular disease resistance in Nicotiana benthamiana (Nakano & Mukaihara, R proteins often carry nucleotide-binding (NB) and leucine-rich re- 2019). Interestingly, Roq1 was originally identified to recognize peat (LRR) domains (Jones & Dangl, 2006; Qi & Innes, 2013). The se- Pseudomonas and Xanthomonas effectors HopQ1 and XopQ, respec- quences of NB-LRR receptor (NLR) proteins are highly polymorphic, tively (Schultink et al., 2017). Importantly, transgenic expression presumably due to a molecular arms race shaped by coevolution with of RPS4/RRS1 or Roq1 in tomato confers bacterial wilt resistance their corresponding Avrs (Baggs et al., 2017; Bergelson et al., 2001). (Narusaka et al., 2013; Thomas et al., 2020). More recently, the Consequently, significant natural variation for NLR-dependent Avr Solanum lycopersicoides NLR protein Ptr1 was shown to recognize effector recognition is often observed within a plant species (Ma RipBN and P. syringae effector AvrRpt2 (Mazo-Molina et al., 2019). et al., 2006; McCann & Guttman, 2008). In addition to these avirulence effectors for which corresponding Ralstonia solanacearum causes bacterial wilt that results in sig- NLRs have been identified, the R. solanacearum effectors AvrA nificant losses during crop cultivation. Bacterial wilt causes severe (RipAA) and RipAX2 act as avirulence proteins that induce bacterial damage in more than 200 plant species, including many solanaceous wilt resistance in tobacco and eggplant, respectively (Morel et al., crops such as tomato, potato, and pepper (Genin & Denny, 2012). 2018; Poueymiro et al., 2009). PopP1 (RipP1) plays a role in aviru- R. solanacearum is a soilborne bacterium, entering host plant roots lence in tobacco and petunia (Chen et al., 2018; Lavie et al., 2002; mostly through natural wounds, and colonizes plant xylem vessels, Poueymiro et al., 2009). Moreover, several R. solanacearum effec- resulting in wilt symptoms (Denny, 2007). R. solanacearum is often tors were shown to induce cell death when transiently expressed in referred to as the Ralstonia solanacearum species complex (RSSC) plant cells, such as AWR1 (RipA1) (Jeon et al., 2020; Prokchorchik due to its extensive genetic diversity (Fegan & Prior, 2005). RSSC is et al., 2020; Sole et al., 2012), AWR2 (RipA2), AWR4 (RipA4), AWR5 classified into four phylotypes based on genomic sequence diversity (RipA5) (Sole et al., 2012), RipE1 (Jeon et al., 2020; Prokchorchik that roughly correlate to geographical distribution: phylotype I to et al., 2020; Sang et al., 2020), RipI (Zhuo et al., 2020), PopA (RipX) Asia, phylotype IIA and IIB to the Americas, phylotype III to Africa, (Arlat et al., 1994; Belbahri et al., 2001; Racape et al., 2005), RipAB and phylotype IV to Australia and Indonesia (Fegan & Prior, 2005). (Zheng et al., 2019), RipAT, RipAV (Clarke et al., 2015; Wroblewski R. solanacearum virulence requires type III secretion system ef- et al., 2009) and RipTPS (Poueymiro et al., 2014). fectors (T3Es) that are translocated into host plant cells during infec- Black nightshades such as Solanum americanum and Solanum ni- tion (Coll & Valls, 2013; Genin & Denny, 2012). Extensive genomic grum are herbaceous plants with a worldwide distribution that are and transcriptomic studies have uncovered the T3E repertoires in thought to have great genetic diversity (Edmonds & Chweya, 1997; the RSSC (Genin & Denny, 2012; Mukaihara et al., 2010; Occhialini Sarkinen et al., 2018). Several studies have shown that black night- et al., 2005; Peeters et al., 2013; Prokchorchik et al., 2020; Sabbagh shades are a useful, underexploited source of disease resistance. For et al., 2019; Valls et al., 2006). In addition, several T3Es from instance, S. nigrum is resistant to tobacco mosaic virus, potato late R. solanacearum have been reported to trigger disease resistance blight, and Phomosis fruit rot (Edmonds & Chweya, 1997). The potato MOON et al. | 319 late blight resistance gene Rpi-amr3 (Resistance to Phytophthora in- reported (Prokchorchik et al., 2020). To identify the candidate aviru- festans) was identified in S. americanum, a possible diploid ancestor lence effectors, effector repertoires of Pe_26 and To_1 strains were of S. nigrum (Ganapathi & Rao, 1987; Witek et al., 2016). compared with the following criteria. First, the five T3Es, RipBA, Identification of a novel avirulence protein is an important step to RipG1, RipTAL, RipY, and RS_T3E_Hyp6, which are present in the develop disease resistance. As black nightshades can be a source of avirulent strain Pe_26 but absent in the virulent To_1, were selected. R genes for other pathogens (Edmonds & Chweya, 1997; Witek et al., Second, the seven T3Es, RipAX1, RipG6, RipAZ1, AWR3 (RipA3), 2016), we hypothesized that some black nightshade accessions might RipB, RipD, and RipJ, which are present in both strains but showing harbour bacterial wilt resistance genes. In this study, we identified less than 95% amino acid identity, were chosen. As a result, a total of R. solanacearum T3E RipAZ1 as an avirulence determinant in 12 candidate avirulence effectors were selected. S. americanum using a bacterial wilt disease assay and comparative Pathogen avirulence proteins often induce cell death when tran- genomics analysis. We showed that the deletion of ripAZ1 in an aviru- siently expressed in plant cells using Agrobacterium-mediated transient lent R. solanacearum strain resulted in bacterial wilt disease symptom transformation (hereafter, agroinfiltration). To identify the avirulence development in a resistant S. americanum accession. We show that effector that induces cell death in S. americanum SP2273 but not cytoplasmic localization may be required for RipAZ1-induced cell SP2275, the 12 selected candidate avirulence effector genes were death in S. americanum. cloned with a C-terminal FLAG tag under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. The R. solanacearum GMI1000 allele of the effector RipA1 with the C-terminal FLAG tag 2 | R E S U LT S was used as a positive control because it induced robust cell death in all tested S. americanum accessions (data not shown), including SP2273 2.1 | S. americanum shows accession-specific and SP2275, when transiently expressed (Figures 2a and S1), as well bacterial wilt resistance as in N. benthamiana (Jeon et al., 2020; Prokchorchik et al., 2020; Sole et al., 2012). C-terminal FLAG-tagged yellow fluorescent protein (YFP) To identify a bacterial wilt resistance source in black nightshades, was expressed as a negative control. Agroinfiltration of each of the 12 we tested 26 S. americanum accessions (Table S1) for disease sus- candidate avirulence effectors in S. americanum SP2275 did not result ceptibility to seven R. solanacearum phylotype I strains isolated from in cell death, in contrast to RipA1GMI1000 (Figure S1). However, agroin- diseased pepper (Pe_1, Pe_13, Pe_26, Pe_57) or tomato (To_1, To_7, filtration of the candidate avirulence effector RipAZ1 induced strong To_63) plants in Korea (Table 1) (Segonzac et al., 2017). S. ameri- cell death in SP2273 (Figure 2a). Next, we quantified the RipAZ1- canum plants were infected with R. solanacearum using the soil- induced S. americanum accession-specific cell death using an electro- drenching method and wilt symptoms were observed at 14 days lyte leakage assay (Figure 2b). As expected, RipA1GMI1000 induced a postinfection (dpi). Among these interactions, we found that R. sola- robust increase in conductivity in SP2273 and SP2275 while YFP did nacearum strains Pe_26 and To_1 showed the most reproducible and not (Figure 2b). Consistent with our cell death assay results (Figure 2a), robust avirulence and virulence phenotypes, respectively, in S. amer- agroinfiltration of RipAZ1 led to increased conductivity, comparable to icanum accession SP2273. Therefore, these two strains were chosen RipA1GMI1000 in S. americanum SP2273 but not in SP2275 (Figure 2b). for further analysis. R. solanacearum Pe_26 caused strong disease This result further demonstrates that RipAZ1-triggered cell death in symptoms in S. americanum SP2275 but not in SP2273 (Figure 1a). S. americanum is accession-specific. The lack of RipAZ1-induced cell In contrast, R. solanacearum To_1 caused comparable disease symp- death in SP2275 was not due to protein instability because transiently toms in both accessions SP2273 and SP2275 (Figure 1a). In addition, expressed RipAZ1-FLAG protein was stable (Figure S2). Our data we performed a quantitative assessment of disease by calculating therefore strongly suggest that RipAZ1 may function as a major aviru- the survival rate of the S. americanum plants infected with R. solan- lence determinant in SP2273. However, we cannot exclude the possi- acearum Pe_26 or To_1 (Figure 1b). Only the S. americanum SP2273 bility that RipTAL, RipY, and RipA3 may have not induced cell death due plants infected with R. solanacearum Pe_26 showed a significantly to the lack of protein expression (Figure S2). high survival rate at 10 dpi (Figure 1b). These data suggest that R. solanacearum Pe_26 may carry an avirulence effector(s) that trig- gers bacterial wilt disease resistance in S. americanum SP2273. 2.3 | RipAZ1 confers R. solanacearum avirulence in S. americanum SP2273 2.2 | R. solanacearum effector RipAZ1 induces We next hypothesized that a loss of function mutation in ripAZ1 accession-specific cell death in S. americanum would enable R. solanacearum Pe_26 to cause disease in the resist- ant S. americanum accession SP2273. A ripAZ1 knockout mutant We hypothesized that an avirulence effector is present in Pe_26 was constructed by replacing the ripAZ1 open reading frame (ORF) but absent or showing significant sequence polymorphism in To_1. with a spectinomycin resistance (SpecR) cassette through homolo- Recently, the genome sequences and T3E repertoires of multiple gous recombination using pRCgg-ΔripAZ1 (Table 1, and Figures R. solanacearum phylotype I strains, including Pe_26 and To_1, were S3a and S4a). The resulting R. solanacearum Pe_26 ΔripAZ1 mutant 320 | MOON et al. TA B L E 1 Bacterial strains and plasmids used in this study Strain or plasmid Relevant characteristics Source or reference Strains Ralstonia solanacearum Pe_1 Wild-type; isolated from a diseased commercial pepper plant in Korea Segonzac et al. (2017) Pe_13 Wild-type; isolated from a diseased commercial pepper plant in Korea Segonzac et al. (2017) Pe_26 Wild-type; isolated from a diseased commercial pepper plant in Korea Segonzac et al. (2017) Pe_26 ΔripAZ1 In-frame deletion of ripAZ1 replaced by spectinomycin resistance (SpecR) cassette This study using pRCgg-ΔripAZ1; SpecR Pe_26 ΔripAZ1 (ripAZ1) ripAZ1 complementation in Pe_26 ΔripAZ1 using pRCKgg-PhB-ripAZ1-FLAG; This study SpecR , kanamycin resistance (KanR) Pe_57 Wild-type; isolated from a diseased commercial pepper plant in Korea Segonzac et al. (2017) To_1 Wild-type; isolated from a diseased commercial tomato plant in Korea Segonzac et al. (2017) To_7 Wild-type; isolated from a diseased commercial tomato plant in Korea Segonzac et al. (2017) To_63 Wild-type; isolated from a diseased commercial tomato plant in Korea Segonzac et al. (2017) Escherichia coli DH5α Wild-type Laboratory collection Agrobacterium tumefaciens AGL1 Wild-type; ampicillin resistance (AmpR) Laboratory collection Plasmids pICH41021 Modified pUC19 vector in which the internal BsaI recognition site is mutated; Laboratory collection AmpR pICH86988 Binary vector carrying CaMV 35S promoter; KanR Weber et al. (2011) pICH86988-ripA1GMI1000 -FLAG ripA1GMI1000 -FLAG cloned in pICH86988; KanR Laboratory collection R pICH86988-YFP-FLAG YFP-FLAG cloned in pICH86988; Kan Laboratory collection pICH86988-FLAG-GFP FLAG-GFP cloned in pICH86988; KanR Laboratory collection R pICH86988-ripAZ1Pe_26-FLAG ripAZ1Pe_26-FLAG cloned in pICH86988; Kan This study pICH86988-ripTAL-FLAG ripTAL-FLAG cloned in pICH86988; KanR This study R pICH86988-ripY-FLAG ripY-FLAG cloned in pICH86988; Kan This study pICH86988-RS_T3E_Hyp6-FLAG RS_T3E_Hyp6-FLAG cloned in pICH86988; KanR This study R pICH86988-ripBA-FLAG ripBA-FLAG cloned in pICH86988; Kan This study pICH86988-ripG1-FLAG ripG1-FLAG cloned in pICH86988; KanR This study R pICH86988-ripAX1-FLAG ripAX1-FLAG cloned in pICH86988; Kan This study pICH86988-ripB-FLAG ripB1-FLAG cloned in pICH86988; KanR This study R pICH86988-ripG6-FLAG ripG6-FLAG cloned in pICH86988; Kan This study pICH86988-ripA3-FLAG ripA3-FLAG cloned in pICH86988; KanR This study R pICH86988-ripD-FLAG ripD-FLAG cloned in pICH86988; Kan This study pICH86988-ripJ-FLAG ripJ-FLAG cloned in pICH86988; KanR This study R pICH86988-ripAZ1Pe_51-FLAG ripAZ1Pe_51-FLAG cloned in pICH86988; Kan This study pICH86988-ripAZ1Pe_57-FLAG ripAZ1Pe_57-FLAG cloned in pICH86988; KanR This study R pICH86988-ripAZ1To_1-FLAG ripAZ1To_1-FLAG cloned in pICH86988; Kan This study pICH86988-ripAZ1SL3022-FLAG ripAZ1SL3022-FLAG cloned in pICH86988; KanR This study R pICH86988-ripAZ1KACC10722-FLAG ripAZ1KACC10722-FLAG cloned in pICH86988; Kan This study pICH86988-ripAZ124-277-FLAG ripAZ124-277-FLAG cloned in pICH86988; KanR This study 59-277 59-277 R pICH86988-ripAZ1 -FLAG ripAZ1 -FLAG cloned in pICH86988; Kan This study pICH86988-ripAZ187-277-FLAG ripAZ187-277-FLAG cloned in pICH86988; KanR This study 1-271 1-271 R pICH86988-ripAZ1 -FLAG ripAZ1 -FLAG cloned in pICH86988; Kan This study (Continues) MOON et al. | 321 TA B L E 1 (Continued) Strain or plasmid Relevant characteristics Source or reference 1-255 1-255 R pICH86988-ripAZ1 -FLAG ripAZ1 -FLAG cloned in pICH86988; Kan This study pICH86988-ripAZ159-271-FLAG ripAZ159-271-FLAG cloned in pICH86988; KanR This study R pICH86988-ripAZ1To_1-YFP ripAZ1To_1-YFP cloned in pICH86988; Kan This study pICH86988-ripAZ1Pe_51-YFP ripAZ1Pe_51-YFP cloned in pICH86988; KanR This study R pICH86988-ripAZ1Pe_51-YFP-NES ripAZ1Pe_51-YFP-NES cloned in pICH86988; Kan This study pICH86988-ripAZ1Pe_51-YFP-NLS ripAZ1Pe_51-YFP-NLS cloned in pICH86988; KanR This study mut mut R pICH86988-ripAZ1Pe_51-YFP-NES ripAZ1Pe_51-YFP-NES cloned in pICH86988; Kan This study pICH86988-ripAZ1Pe_51-YFP-NLSmut ripAZ1Pe_51-YFP-NLSmut cloned in pICH86988; KanR This study R pICH86988-ripAZ1Pe_26-YFP ripAZ1Pe_26-YFP cloned in pICH86988; Kan This study pRCgg 5′-BamHI-BsaI-lacZ-BsaI-EcoRI-3′ fragment cloned in pICH41021; AmpR This study pRCKgg Modified pRCK vector (Monteiro et al., 2012) in which Gateway cassette is This study replaced with Golden Gate cassette (lacZ); AmpR , KanR pRCgg-ΔripAZ1 SpecR cassette from pCR8, flanked by 5′ and 3′ sequences of ripAZ1Pe_26 ORF, This study cloned in pRCgg; AmpR , SpecR pRCKgg-PhB-ripAZ1-FLAG ripAZ1Pe_26-FLAG under the control of hrpB promoter (PhB) cloned in pRCKgg; This study AmpR , KanR strain was experimentally validated to carry the desired mutation of RipAZ1, we used the publicly available database RalstoT3E (Figure S4b). To test if RipAZ1 is required for R. solanacearum Pe_26 (https://iant.toulo​use.inra.fr/T3E from Peeters et al., 2013) and the avirulence, the resistant and susceptible S. americanum accessions R. solanacearum phylotype I strain database from Prokchorchik et al. were infected with wild-type or ΔripAZ1 mutant Pe_26 strain using (2020) to identify 21 nonredundant RipAZ1 natural sequence vari- the soil-drenching infection assay. As expected, wild-type R. sola- ants (Table S2 and Figure S6). Phylogenetic analysis indicated that nacearum Pe_26 caused severe wilt symptoms in S. americanum the RipAZ1 amino acid sequence of R. solanacearum phylotype IV SP2275, whereas SP2273 remained healthy (Figure 3a). In contrast, strain SL3022 is the most distantly related to RipAZ1Pe_26 (Figure R. solanacearum Pe_26 ΔripAZ1 caused comparable wilt symp- S6). Sequences of RipAZ1 natural variants from the same phylotype toms in both S. americanum accessions (Figure 3a). Importantly, the showed more than 80% amino acid sequence identity (Figures 4a complemented strain Pe_26 ΔripAZ1 carrying ripAZ1, constructed and S7). The C-terminal region of pathogen effectors is often criti- using pRCKgg-PhB-ripAZ1-FLAG (Table 1, and Figures S3b and S5), cal for function (Bos et al., 2006; Catanzariti et al., 2010; Yang et al., showed S. americanum SP2273-specific avirulence, demonstrating 2005; Yang & White, 2004). Interestingly, ripAZ1To_1 carries a one that RipAZ1 is the avirulence determinant (Figure 3a). We further nucleotide base pair (bp) insertion that causes a frameshift and assessed the survival rate of S. americanum plants infected with wild- C-terminal truncation of 22 amino acids (Figures 4a and S7). In ad- type, ΔripAZ1, or ΔripAZ1 (ripAZ1) strains of R. solanacearum Pe_26 dition, RipAZ1 variants of phylotype IV strains carry 6-amino acid (Figure 3b). None of the S. americanum SP2275 plants infected by extended C-termini. Therefore, four representative phylotype I and Pe_26 ΔripAZ1 mutant strain survived at 10 dpi, suggesting that the two of the most distantly related phylotype IV variants were selected loss of ripAZ1 does not result in a significant decrease in virulence for further analysis (Figures 4a and S6). To compare the avirulence of R. solanacearum Pe_26 in the susceptible host plants (Figure 3b). of RipAZ1 natural variants, we used an agroinfiltration assay in the In contrast, the S. americanum SP2273 plants showed a significantly resistant S. americanum accession SP2273. As previously shown, higher survival rate when infected with R. solanacearum Pe_26 agroinfiltration of RipA1GMI1000 or the RipAZ1Pe_26 induced strong wild-type or ΔripAZ1 (ripAZ1) strain as compared to To_1 or Pe_26 cell death, whereas YFP did not (Figure 4b). Importantly, agroinfiltra- ΔripAZ1 strain (Figure 3b). These results demonstrate that RipAZ1 tion of RipAZ1 natural variants except RipAZ1To_1 induced strong cell confers R. solanacearum Pe_26 avirulence in S. americanum. death and high conductivity in S. americanum SP2273 (Figures 4b and S8, and Table S3). We found that RipAZ1To_1 protein accumulated to a similar level to other variants, indicating that the lack of cell death 2.4 | The C-terminally truncated RipAZ1 natural of RipAZ1To_1 is not due to protein instability (Figure 4c). These data variant is unable to induce cell death in S. americanum indicate that RipAZ1 avirulence is conserved across different phylo- types and that the C-terminal region is indispensable for recognition. Naturally occurring avirulence effectors often show significant se- Because RipAZ1 cannot induce cell death in N. benthamiana, we set quence polymorphism in pathogen populations (Asai et al., 2018; out an experiment to test its ability to interfere with plant defence re- Dodds et al., 2006). To survey natural sequence polymorphisms sponses. We examined the amplitude of reactive oxygen species (ROS) 322 | MOON et al. F I G U R E 1 Ralstonia solanacearum strain Pe_26 induces accession-specific disease resistance in Solanum americanum. Bacterial wilt symptoms (a) and a survival graph (b) of the S. americanum accessions SP2273 and SP2275 infected with wild-type R. solanacearum strains Pe_26 and To_1. (a) Photographs from representative plants were taken at 14 days postinoculation. Scale bar = 5 cm. (b) The percentage of surviving plants was recorded for 10 days. The data used for the survival graph were collected from three independent experiments. Log- rank (Mantel–Cox) test p values are .0001 and .373 in SP2273 and SP2275, respectively production and defence gene expression triggered by bacterial flagel- truncated natural variants and presence/absence polymorphism. lin peptide flg22 in N. benthamiana leaves expressing YFP, RipAZ1Pe_26, RipAZ1 variants with C-terminal truncation were identified in only 33 or RipAZ1To_1 (Figure S9). We observed a significant reduction of the R. solanacearum phylotype I strains (Table S2). Eighty strains across total flg22-induced ROS production in the presence of RipAZ1Pe_26 phylotypes carried full-length (255 amino acids) or C-terminally ex- but not of RipAZ1To_1 compared to the sample expressing YFP (Figure tended (284 amino acids) variants of the RipAZ1 sequence (Table S2). S9a). Similarly, the induction of NbACRE31 expression by flg22 was Interestingly, the C-terminally extended RipAZ1 sequence was significantly reduced in leaves expressing RipAZ1Pe_26 compared to found in much higher frequency in phylotype IV strains (23 out of 27 RipAZ1To_1 or YFP (Figure S9b). As both RipAZ1 variants accumulated surveyed) compared to other phylotypes. These data further sup- to a similar level in N. benthamiana cells (Figure S9c), these data indicate port a critical role for the C-terminal region of RipAZ1. that RipAZ1could modulate the host defence responses and that the RipAZ1 C-terminal region might contribute to this function. To survey the distribution of RipAZ1 in RSSC, we collected 2.5 | The truncated variant RipAZ159-271 induces cell RipAZ1 sequence information from publicly available databases death in S. americanum (Peeters et al., 2013; Prokchorchik et al., 2020). RipAZ1 was distrib- uted in all four phylotypes among the 168 RSSC strains surveyed. To determine the minimal region required for RipAZ1 function, five N- Because R. solanacearum RipAZ1To_1 was the only experimentally or C-terminally truncated RipAZ1Pe_51 variants were generated based on validated virulent variant, we focused on identifying C-terminally the predicted secondary structure of the protein (Figures 5a and S10). MOON et al. | 323 F I G U R E 2 Overexpression of RipAZ1 induces cell death in Solanum americanum SP2273. (a) The candidate avirulence effectors were agroinfiltrated in SP2273 leaf epidermal cells. RipA1GMI1000 and yellow fluorescent protein (YFP) were used as controls. Photographs were taken at 2 days postinfiltration. White circles indicate the infected leaf area showing cell death. (b) Conductivity measurement of RipAZ1Pe_26 in SP2273 and SP2275. Four-week-old S. americanum leaves transiently expressing RipAZ1Pe_26 or control proteins, RipA1GMI1000 and YFP, using agroinfiltration were taken for conductivity measurements. HPI indicates hours postinfiltration. The data used were collected from three independent experiments. Coloured shapes and grey lines indicate means and standard deviations, respectively. Different letters indicate groups showing statistically significant differences at 48 HPI (p < .05; Student's t test). p values of circle–triangle, triangle–square, and square–circle are as follows: 0.343, 6.88 × 10−11, and 2.33 × 10−11 in SP2273; 2.20 × 10−16, 0.885, and 4.20 × 10−16 in SP2275 Agroinfiltration of the truncated RipAZ1 Pe_51 variants did not lead to 2.6 | RipAZ1-YFP localizes in the cell death in S. americanum SP2273 when N-terminal 86 (87–277) or nucleus and cytoplasm when transiently expressed in C-terminal 22 (1–255) amino acids were absent (Figures 5b and S11, N. benthamiana leaf epidermal cells and Table S3). However, the loss of 58 N-terminal amino acids (59–277) or six C-terminal amino acids (1–271) did not affect RipAZ1-induced To examine RipAZ1 localization in plant cells, C-terminally YFP- cell death (Figures 5b and S11, and Table S3). We next tested the tagged RipAZ1 variants were transiently expressed in N. benthami- RipAZ159-271 variant in which 58 N-terminal and six C-terminal amino ana. Agroinfiltration of RipAZ1-YFP triggered cell death comparable acids are truncated. Agroinfiltration of RipAZ159-271 induced strong to RipAZ1-FLAG in S. americanum SP2273, indicating that RipAZ1- cell death that was comparable to that induced by full-length RipAZ1 YFP is functional (Figure S12). RipAZ1Pe_26-YFP and RipAZ1Pe_51- (1–277) (Figures 5b and S11). Immunoblot analysis showed that all YFP, which induce cell death in SP2273, were both localized in RipAZ1 variants were expressed, indicating that the loss of cell death- the nucleus and cytoplasm of N. benthamiana leaf epidermal cells inducing activity in some truncated RipAZ1 variants was not due to pro- (Figure S13). Similarly, RipAZ1To_1-YFP, which does not trigger cell tein instability (Figure 5c). Our results demonstrate that the centrally death in SP2273, also localized in the nucleus and cytoplasm when located region containing 213 amino acids is sufficient for RipAZ1 aviru- expressed in N. benthamiana leaves (Figure 6a). To investigate the lence and that the predicted α-helices B and G (Figure S10) may be re- contribution of RipAZ1 localization to its avirulence, we generated quired for the avirulence. RipAZ1Pe_51-YFP variants that are translationally fused to a nuclear 324 | MOON et al. F I G U R E 3 RipAZ1 triggers accession-specific immune responses in Solanum americanum. Bacterial wilt symptoms (a) and a survival graph (b) of accessions SP2273 and SP2275 infected with wild-type, ΔripAZ1, or ΔripAZ1 (ripAZ1) strain of Pe_26. (a) Photographs from representative plants were taken at 10 days postinfiltration. Scale bar = 5 cm. (b) The percentage of survival plants was recorded for 10–20 days. The data used for survival graphs were collected from three independent experiments. Log-rank (Mantel–Cox) test p values are <.0001 and .128 in SP2273 and SP2275, respectively export signal (NES) or nuclear localization signal (NLS) (Heidrich still showed the nucleocytoplasmic localization (Figure 6a). Next, et al., 2011; Wen et al., 1995). Nonfunctional variants of NES and we agroinfiltrated the RipAZ1-YFP variants in S. americanum NLS, NESmut and NLSmut , respectively, were used as controls. An SP2273 to test their cell death-inducing activity. Surprisingly, both immunoblot assay using anti-GFP antibody confirmed the stable RipAZ1Pe_51-YFP-NES and RipAZ1Pe_51-YFP-NLS variants induced expression of all RipAZ1-YFP variants in N. benthamiana leaf cells cell death comparable to RipAZ1Pe_51-YFP (Figures 6b and S15, and (Figure S14). RipAZ1Pe_51-YFP-NES or RipAZ1Pe_51-YFP-NLS mainly Table S3). However, we favour the hypothesis that RipAZ1 recogni- localized to the cytoplasm or nucleus, respectively (Figure 6a). As tion occurs in the cytoplasm because RipAZ1Pe_51-YFP-NLS could expected, RipAZ1Pe_51-YFP-NESmut and RipAZ1Pe_51-YFP-NLSmut still induce defence responses prior to localization in the nucleus. MOON et al. | 325 F I G U R E 4 A naturally occurring C-terminally truncated RipAZ1 variant does not induce cell death in Solanum americanum. Amino acid sequence comparison (a), cell death responses in SP2273 (b), and western blot analysis in Nicotiana benthamiana (c) of the six representative RipAZ1 natural variants selected for functional analysis. (a) Nonsynonymous single nucleotide polymorphisms are indicated as black lines. A grey box indicates the region with no sequence identity due to a frameshift mutation caused by 1-base pair insertion as indicated with an asterisk. An amino acid in-frame-insertion is indicated with black triangles. Phylotype is abbreviated as “Phy”. (b) The photograph was taken at 2 days postinfiltration. White circles indicate the infected leaf area showing cell death. (c) Empty vector (EV; pICH86988) and yellow fluorescent protein (YFP)-FLAG were used as controls. Proteins were immunoblotted with anti-FLAG antibody. Leaf tissue was harvested at 2 days postinfiltration. Ponceau staining shows equal loading of proteins 3 | D I S CU S S I O N accession-specific avirulence of R. solanacearum Pe_26 indicates that RipAZ1-induced cell death is more likely to be an ETI response We show here that the effector RipAZ1 from the bacterial wilt- than due to effector toxicity. Moreover, we identified the minimal causing pathogen R. solanacearum triggers immunity in the wild region of RipAZ1 protein as RipAZ159-271 that is sufficient to acti- Solanaceae species S. americanum SP2273. The S. americanum vate defence. Based on this result, RipAZ1 N-terminus, including a 326 | MOON et al. F I G U R E 5 Analysis of truncated RipAZ1 defines the minimal region sufficient for cell death-inducing activity. Schematics (a), cell death responses in SP2273 (b), and western blot analysis in Nicotiana benthamiana (c) of truncated variants of RipAZ1Pe_51-FLAG. (a) The N- or C-terminally truncated variants of RipAZ1Pe_51 were based on the predicted secondary structure (Figure S10). Numbers indicate amino acids. Variants inducing cell death in SP2273 are indicated on the right panel with “+”. (b) The photograph was taken at 2 days postinfiltration. White circles indicate the infected leaf area showing cell death. (c) Empty vector (EV; pICH86988) and YFP-FLAG were used as controls. Proteins were immunoblotted with anti- FLAG antibody. Leaf tissue was harvested at 2 days postinfiltration. The protein bands with expected molecular weight are indicated with white asterisks. Ponceau staining shows equal loading of proteins disordered region (first to 42nd amino acids) and the first α-helix αA for avirulence did not affect RipAZ1 avirulence. Despite our (Figure S10), is not required for the recognition. As the N-terminus efforts to predict biochemical function of RipAZ1 based on the is predicted to be a disordered region, it may contain a type III se- secondary structure, we were unable to obtain a meaningful re- cretion signal similar to many other T3Es (Guttman & Greenberg, sult. To get insights into RipAZ1 biochemical function, it will be 2001; Mudgett & Staskawicz, 1999). Further C-terminal truncation important to investigate its tertiary structure in future. from RipAZ159-271 led to the loss of its recognition. Based on our The Arabidopsis NLR protein RRS1 localizes exclusively in the nu- survey and result with RipAZ1To_1, RipAZ1 natural variants that cleus when PopP2 is present (Deslandes et al., 2003). Subsequently, may have lost avirulence due to frameshift mutations were found PopP2 acetylates and reconfigures RRS1 in the plant cell nucleus and (Table S2 and Figure 4). This may be an indication of the evolution- activates RPS4-dependent immunity (Guo et al., 2020; Le Roux et al., ary pressure for R. solanacearum to evade recognition of RipAZ1 in 2015; Sarris et al., 2015). Interestingly, it was also shown that the nature. Interestingly, C-terminal extension (279th to 284th amino Arabidopsis cysteine protease RD19 (Response to Dehydration 19), acids) of RipAZ1 natural variants in phylotype IV strains main- which is required for RRS1-dependent immunity, relocalizes to the tained the cell death-inducing activity (Figure 4b). Furthermore, plant nucleus in the presence of PopP2 (Bernoux et al., 2008). In con- based on our survey of RipAZ1 polymorphisms in multiple R. sola- trast to PopP2, the forced localization of RipAZ1 to the cytoplasm nacearum strains (Figure 4a), many nonsynonymous single nucleo- did not result in loss of avirulence function. Based on this observa- tide polymorphisms (SNPs) found in the minimal region sufficient tion, it is reasonable to hypothesize that the corresponding R protein MOON et al. | 327 unlikely that a similar mechanism exists for RipAZ1 because forced localization in the nucleus or cytoplasm did not affect cell death-in- ducing activity. Taken together, identification of the corresponding R protein and/or the direct target would significantly help elucidate the molecular basis of RipAZ1-triggered defence activation. Identification of avirulence proteins secreted by pathogens is an important step towards effector-assisted molecular breeding for enhanced disease resistance (Jayaraman et al., 2016; Vleeshouwers & Oliver, 2014). Our finding that RipAZ1 functions as an avirulence determinant of the R. solanacearum–S. americanum interaction sug- gests a possibility of identifying a novel bacterial wilt resistance gene using RipAZ1-induced cell death as a proxy for a disease resistance phenotype. Recognition of avirulence effectors by corresponding NLR proteins generally results in the activation of strong defence responses and restricts pathogen proliferation. However, pathogens often overcome the plant defence through modification of the effec- tor repertoire. A bacterial spot pathogen Xanthomonas euvesicatoria T3E protein AvrBs2 is known to be highly conserved because it is required for full pathogen virulence (Gassmann et al., 2000; Kearney & Staskawicz, 1990). Hence, it would be necessary to test if RipAZ1 is required for full virulence of R. solanacearum. Of note, our tran- sient assay results in N. benthamiana suggest that RipAZ1 could mod- ulate host defence responses (Figure S9). Hence, it is plausible that RipAZ1 contributes to the full virulence of R. solanacearum. If RipAZ1 is essential for pathogen virulence, the corresponding R gene may be used to develop durable disease resistance in the future. Pathogens are under constant evolutionary pressure to evade recognition. As a result, loss-of-function mutations are often iden- tified in avirulence effectors (Gassmann et al., 2000; Jones & Dangl, 2006). Therefore, introducing multiple NLRs and pattern-recognition receptors (PRRs) with distinct ligand recognition specificities in a crop cultivar is desirable to achieve durable disease resistance. R gene pyr- amiding would be particularly ideal for developing durable disease F I G U R E 6 RipAZ1-YFP localizes in the nucleus and cytoplasm resistance to R. solanacearum due to its highly polymorphic T3E rep- when transiently expressed in Nicotiana benthamiana leaf epidermal ertoires (Fukuoka et al., 2015). The Arabidopsis PRR elongation fac- cells. Subcellular localization in N. benthamiana (a) and cell death tor-Tu (EF-Tu) receptor (EFR) detects bacterial EF-Tu and activates responses in SP2273 (b) of RipAZ1-YFP variants when transiently antibacterial immunity (Jones & Dangl, 2006; Lacombe et al., 2010). expressed using agroinfiltration. (a) YFP signals were observed at Significantly, the interfamily transfer of EFR has resulted in enhanced 2 days postinfiltration. Scale bar = 50 μm. (b) The photograph was taken at 2 days postinfiltration bacterial wilt resistance in several plant species, including Solanum lycopersicum, Solanum commersonii, N. benthamiana, and Medicago capable of recognizing RipAZ1 functions in the cytoplasm and nu- truncatula (Boschi et al., 2017; Lacombe et al., 2010; Pfeilmeier et al., cleus. Alternatively, a guardee/decoy protein targeted by RipAZ1 2019). In addition to the aforementioned NLRs such as RPS4/RRS, could relocalize to the subcellular compartment where the corre- Roq1, and Ptr1, identification of an additional R protein that recog- sponding R protein is present. For example, the 50 kDa helicase (p50) nizes RipAZ1 would be helpful in developing durable bacterial wilt domain of tobacco mosaic virus interacts with N receptor-interacting resistance. If successfully identified in the future, deployment of the protein NRIP1 and induces its localization to the cytoplasm and nu- RipAZ1-recognizing R gene would be highly valuable particularly with cleus to activate N protein-dependent immunity (Caplan et al., 2008). Roq1 when introduced in a crop species because the two R genes are Previously, many pathogen effectors were shown to localize to both predicted to confer resistance to more than 80% of R. solanacearum nucleus and cytoplasm in plant cells (Choi et al., 2017). In addition strains based on our survey results (Table S2) and the RalstoT3E to the aforementioned PopP2, P. syringae T3E AvrRps4 localized in database (Peeters et al., 2013). However, we expect that the plant cell nucleus and cytoplasm is recognized by RPS4/RRS1 paired S. americanum R gene recognizing RipAZ1 alone may also be used NLR proteins. Interestingly, AvrRps4 induces cell compartment- to develop bacterial wilt-resistant crops. In particular, the RipAZ1- specific defence responses (Heidrich et al., 2011). However, it seems recognizing R gene could be most useful in conferring bacterial wilt 328 | MOON et al. resistance against R. solanacearum phylotype IV where more than with 10 mM MgCl2 . Roots were artificially wounded with a 85% of strains seem to carry functional ripAZ1 (Table S2). Moreover, scalpel and the soil in each pot was drenched with 15 ml of the the RipAZ1 recognition property might be particularly useful for de- diluted bacterial suspension. Plants were then incubated for veloping a bacterial wilt-resistant eggplant cultivar. Sabbagh et al. 10–20 days in the controlled growth room for observation of dis- (2019) have reported that all the strains isolated from eggplant (n = 9) ease symptoms. carry ripAZ1. Consistently, 10 out of 11 strains isolated from egg- plant in our survey (Table S2) seem to carry functional ripAZ1. Witek et al. (2016) have shown that transgenic potato plants expressing the 4.4 | Survival analysis for disease quantification S. americanum NLR Rpi-amr3 are resistant to P. infestans. Because R. solanacearum is one of the major pathogens of potato, identifi- Plants were infected with R. solanacearum strains using the soil- cation of the S. americanum R gene recognizing RipAZ1 may help to drenching infection assay. A total of 24 plants were tested for each develop a potato variety that is resistant to both P. infestans and R. so- strain. The number of surviving plants was counted on a daily basis. lanacearum in the future. Plants with complete wilting were considered dead (Lonjon et al., 2017). The percentage of survival was plotted using the Kaplan– Meier survival analysis with log-rank (Mantel–Cox) test. p values 4 | E X PE R I M E NTA L PRO C E D U R E S smaller than 0.05 indicate significant differences among survival curves of the tested strains. Graphs and statistical analyses were 4.1 | Plant materials and growth conditions generated with Prism v. 8.4.3 (GraphPad Software Inc.). S. americanum accessions used in this study were obtained from The Sainsbury Laboratory (Norwich, UK) and are indicated in Table S1. 4.5 | Agrobacterium-mediated transient S. americanum seeds were placed on a wet tissue and incubated at transformation 28/18 °C for 16 /8 hr, respectively, with 70% humidity for 1 week for germination. Seedlings were then transferred to individual cell trays Binary constructs (pICH86988 and its derivatives; Table 1) were and grown for 4 weeks in a controlled room at 24 °C with 16 hr of introduced into A. tumefaciens AGL1 by electroporation and the light a day. N. benthamiana seeds were sown, germinated, and grown resulting transformants were selected on low salt Luria (L) agar me- in individual pots for 4–6 weeks in the controlled growth room with dium containing kanamycin (50 µg/ml). Each A. tumefaciens AGL1 the same conditions. transformant was cultured in 3 ml of L medium supplemented with kanamycin (50 µg/ml) for 2 days at 28 °C with shaking (200 rpm). The cultured cells harbouring pICH86988 or its derivative were 4.2 | Bacterial strains and culture conditions harvested by centrifugation and resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES adjusted to pH 5.6 with KOH). The opti- The bacterial strains used in this study are indicated in Table 1. cal density of the suspension was adjusted to OD600 0.4. The bac- Cloning of constructs was carried out in Escherichia coli DH5α. terial suspension was infiltrated into the abaxial side of 4-week-old Transient expression assay for macroscopic cell death, western blot, leaves using a blunt-end needless syringe. Macroscopic cell death and confocal microscopy was carried out using Agrobacterium tume- was observed and photographed at 2–4 days postinfiltration. For faciens AGL1. Soil-drenching infection was achieved using R. sola- protein expression analysis, A. tumefaciens AGL1 harbouring P19, a nacearum strains. E. coli and A. tumefaciens strains were grown in viral silencing suppressor, was coexpressed with all samples after its liquid Luria medium supplemented with appropriate antibiotics for optical density was adjusted to OD600 0.1. 24 hr at 37 and 28 °C, respectively. R. solanacearum strains were grown on peptone broth medium (1% Bacto peptone, 0.1% yeast extract, 0.1% casamino acid) for genomic DNA extraction and natu- 4.6 | Conductivity measurement ral transformation, or BG complete medium (peptone broth medium supplemented with 0.005% triphenyl tetrazolium chloride [TTC], Following Agrobacterium-mediated transient transformation, three 1.5% Bacto agar, and 0.5% glucose) for soil-drenching infection, leaf discs of 9 mm diameter were harvested, transferred to 2 ml of with appropriate antibiotics for 48 hr at 28 °C (Boucher et al., 1985). fresh water, and incubated at room temperature for 2 hr with mild shaking (50–100 rpm). The conductivities of three technical repli- cates per experiment were measured right after infiltration (0 hpi) 4.3 | Soil-drenching infection and every 24 hr until 48 or 72 hpi with a LAQUAtwin EC-33 con- ductivity meter (Horiba). The conductivity measurements of biologi- R. solanacearum cells were scraped off the growth medium plates cal repeats were pooled and the average value is shown. Two-tailed with 10 mM MgCl2 and collected in conical tubes. The bacterial Student's t test was conducted to provide groups showing statisti- suspension was diluted to OD600 0.5 (approximately 5 × 10 8 cfu/ml) cally significant differences (p < .05). MOON et al. | 329 4.7 | Preparation and natural transformation of lacZ cassette amplified from pICH86988 (Weber et al., 2011) was R. solanacearum competent cells prepared as a pICH41021 module construct, also flanked by BsaI recognition sites. Six modules (GMI-1, Tfd terminators, kanamy- The protocol of Perrier et al. (2018) was followed with minor modifi- cin resistance cassette, lacZ cassette for Golden Gate cloning, Tt7 cations. A single colony of R. solanacearum was inoculated into 20 ml terminator, and GMI-2) were then assembled in the pRCgg plasmid of one-quarter strength M63 minimal medium (15 mM ammonium (Figure S3a), converting pRCK to pRCKgg (Table 1 and Figure S3b). sulphate, 1.8 µM iron(II) sulphate, 100 mM monopotassium phos- phate, and 1 mM magnesium sulphate adjusted to pH 7 with KOH) supplemented with 2% glycerol (Miller, 1992) and incubated over- 4.9 | Plasmid construction night at 28 °C with shaking (200 rpm). From 500 ng to 200 µg of plasmid DNA for integration into the bacterial genome was prepared Effector constructs were cloned using the Golden Gate cloning sys- and linearized using SfiI, which does not cut between the two homol- tem as explained by Engler et al. (2008). Genomic DNA of R. sola- ogous recombination sequences. The linearized plasmid constructs nacearum Pe_26, Pe_51, Pe_57, To_1, SL3022, and KACC10722 were purified using a Sepharose 4B column. Bacterial cells were har- was extracted using the Wizard Genomic DNA Purification Kit vested by centrifugation at 6,000 × g for 5 min when the culture (Promega) following the manufacturer's instructions to be used as reached OD600 0.4 to 0.6. The volume of the cells to be collected a PCR template. Gene fragments for some effectors (ripAZ1, ripTAL, was proportional to the amount of the plasmid material (1:10 vol/wt). ripY, and RS_T3E_Hyp6) were amplified from the R. solanacearum The purified DNA was added to the bacterial cells and gently mixed genomic DNA using standard PCR conditions to have BsaI recog- with a pipette. The mixture was spotted on a peptone broth agar me- nition sites at the 5′ and 3′ ends. To construct RipAZ1 truncated dium supplemented with 1.5% Bacto agar and incubated for 2 days variants (Table 1), ATG was additionally included following the BsaI at 28 °C. Grown cells were collected using a sterile spreader and recognition site in forward primers. To construct C-terminal epitope transferred to a new peptone broth agar medium supplemented with tag YFP-NES, YFP-NLS, YFP-NESmut, and YFP-NLSmut modules, appropriate antibiotics. The agar medium was incubated at 28 °C DNA fragments encoding the four aforementioned epitope tags for 2–3 days. The selected colonies were streaked on a new BG were amplified from pICH86988-YFP using standard PCR condi- complete medium supplemented with the antibiotics and colony tions to have NES, NLS, NESmut, or NLSmut at the 3′ end (NES: morphology was confirmed using TTC for its virulence. LALKLAGLDI; NESmut: LALKAAGADA; NLS: PKKKRKVGG; NLSmut: PKTKRKVGG) (Heidrich et al., 2011; Wen et al., 1995). All the am- plified DNA fragments were cloned in SmaI-treated pICH41021 4.8 | pRCgg plasmid vector construction shuttle vector (Table 1). Gene fragments for other effectors (ripBA, ripG1, ripAX1, ripB, ripG6, ripA3, ripD, and ripJ) were synthesized with The pRC (the Ralstonia chromosome) vector series is a powerful tool their codons optimized for synthesis (codon-optimized sequences developed by Monteiro et al. (2012) that can integrate a gene frag- are available upon request) and attached with BsaI recognition sites ment of interest (GOI) cloned using the Gateway cloning system into at the 5′ and 3′ ends by Integrated DNA Technologies (IDT). The an R. solanacearum chromosomal site through a double recombina- synthesized gene fragments were cloned in pICH41021 using SmaI tion event. Here, pRCgg plasmid vectors were generated to trans- (Table 1) by IDT. DNA sequences of these constructs were verified form GOIs into R. solanacearum genomes using the Golden Gate by sequencing using M13F and M13R primers performed by Solgent. cloning system. pICH41021 was used as a backbone plasmid for Gene modules cloned in pICH41021 were assembled using BsaI into pRCgg (Table 1). The lacZ cassette was amplified using standard PCR pICH86988 or pRCKgg with an appropriate promoter and/or appro- conditions to contain BamHI and EcoRI recognition sequences at priate C-terminal epitope tags (Table 1 and Figure S3b). Assembled 5′ and 3′ ends, respectively, followed by BsaI recognition sequences constructs were selected with appropriate antibiotics of the destina- at both ends. BamHI and EcoRI sites within the cassette were then tion vectors and screened with blue-white selection using 15 µl of removed by site-directed mutagenesis. The 5′-BamHI-BsaI-lacZ-BsaI- 100 mM IPTG and 800 µg X-gal. The constructs were finally verified EcoRI-3′ fragment was ligated into pICH41021 with conventional re- by restriction enzyme digestion, followed by visualization in agarose striction enzyme cloning to make the pRCgg plasmid (Table 1 and gel electrophoresis. Figure S3A). To obtain the ripAZ1 knockout plasmid following Wu et al. (2018), Next, GMI-1 and GMI-2, which are conserved genomic regions 1,000 bp of 5′ sequence of the ripAZ1 ORF and 972 bp consisting of used for homologous recombination, transcriptional terminators (Tfd 78 bp of ripAZ1 ORF sequence from its 3′ end and 894 bp of its 3′ and Tt7 terminators), and kanamycin resistance cassette were sep- sequence was amplified from R. solanacearum Pe_26 genomic DNA to arately amplified from pRCK (Monteiro et al., 2012) and prepared have BsaI at both 5′ and 3′ ends and cloned in pICH41021 to be homol- as Golden Gate module constructs in pICH41021, flanked by BsaI ogous recombination fragments. A SpecR cassette amplified from pCR8 recognition sites (Engler et al., 2008). Two BsaI sites within GMI-2 (Invitrogen) flanked by BsaI at both ends was also cloned in pICH41021. were removed by site-directed mutagenesis for further vector con- These modules were assembled using BsaI into pRCgg plasmid (Table 1 struction. To replace the Gateway cassette in pRC vector, another and Figure S3a) and selected with ampicillin and spectinomycin. The 330 | MOON et al. assembled pRCgg-ΔripAZ1 (Table 1) was verified by restriction enzyme NbEF1α, 5′-AGGTCCAGTATGCCTGGGTGCTTGAC-3′ and 5′-AAG digestion, followed by visualization in agarose gel electrophoresis. AATTCACAGGGACAGTTCCAATACCAC-3′; NbCYP71D20, 5′-AAGG TCCACCGCACCATGTCCTTAGAG-3′ and 5′-AAGAATTCCTTGCC CCTTGAGTACTTGC-3′; NbACRE31, 5′-AAGGTCCCGTCTTCGTC 4.10 | Total protein extraction from plant GGATCTTCG-3′ and 5′-AAGAATTCGGCCATCGTGATCTTGGTC-3′ tissues and western blot (Segonzac et al., 2011). Plant tissues were harvested at 2–3 dpi and immediately snap-frozen in liquid nitrogen. The harvested samples were ground and mixed 4.13 | Protein secondary structure prediction with 250 µl of 5 × SDS sample buffer (250 mM Tris-HCl adjusted to pH 6.8, 10% sodium dodecyl sulphate [SDS], 0.1% [wt/vol] bromo- The secondary structure of RipAZ1 was modelled using various phenol blue, and 40% glycerol, supplemented with 50 mM dithi- programs, LRRfinder (Offord et al., 2010), PSIPRED (Buchan & othreitol before use). The mixture was boiled at 96 °C for 10 min, Jones, 2019; Jones, 1999), InterProScan (Jones et al., 2014), and a vortexed vigorously, and centrifuged at 15,000 × g for 1 min. Fifteen plug-in EMBOSS Garnier (Rice et al., 2000) for Geneious v. 11.1.5 microlitres of the supernatant was used for SDS-polyacrylamide gel (Biomatters, Auckland, New Zealand). electrophoresis (PAGE) to separate proteins. Samples were run on an SDS-polyacrylamide gel with 120 V for 80 min, electroblotted onto a PVDF membrane with 300 mA for 120 min and probed with anti- 4.14 | Confocal microscopy FLAG (Sigma-Aldrich) and anti-mouse antibody conjugated to horse- radish peroxidase (HRP) (Sigma-Aldrich) or with HRP-conjugated A. tumefaciens AGL1 carrying C-terminal YFP-tagged ripAZ1 with or anti-GFP (Santa Cruz). Visualization was achieved using SuperSignal without various localization signals was infiltrated in N. benthamiana West Pico and Femto substrate (Thermo Scientific) in ImageQuant leaves. Leaf discs were taken at 2 days postinfiltration from the in- LAS 500 chemiluminescence CCD camera (GE Healthcare). Lastly, filtrated area and mounted in water for confocal laser scanning mi- the membrane was stained with Ponceau S (Sigma-Aldrich) for croscopy. YFP signals were monitored in a confocal laser scanning 10 min to visualize total protein loading. microscope (FV3000, Olympus). The signals were excited with a 488 nm ray line of the argon laser and detected in a confocal channel in the 500–600 nm emission range, applied with a photomultiplier 4.11 | Measurement of ROS production voltage 483 V. Images were obtained using the manufacturer's soft- ware Fluoview FV31S-SW (Olympus). Leaf discs of 5 mm diameter were collected from N. benthamiana leaf tissue expressing RipAZ1Pe_26, RipAZ1To_1, or YFP and were floated AC K N OW L E D G E M E N T S on 150 μl of deionized water overnight. The water was replaced with We thank Dr Heejung Cho and Dr Young Kee Lee for R. solan- 100 μl of solution containing 100 μM luminol (Sigma-Aldrich), 2 μg acearum strains and Dr Jay Jayaraman for critical reading of the of peroxidase from horseradish (Sigma-Aldrich), and 50 nM of flg22 manuscript. This work was supported by a grant from the Next- (Peptron). Luminescence was measured for 75 min using GloMax 96 Generation BioGreen 21 Program (Plant Molecular Breeding microplate luminometer (Promega) and data were recorded as total Center no. PJ01317501), the Rural Development Administration counts of relative light unit (RLU). and National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (NRF-2019R1A2C2084705 & 2018R1A5A1023599 (SRC)), Republic of Korea. C.S. was supported 4.12 | Quantitative reverse transcription PCR by the Creative-Pioneering Researchers Program through Seoul National University. M.V.’s research was funded by the research Two leaf discs of 8 mm diameter were collected from N. bentha- grants (PID2019-108595RB-I00 and SEV/2015/0533) from the miana leaf tissue expressing RipAZ1Pe_26, RipAZ1To_1, or YFP at Spanish Government and the CERCA Program from the Catalan 1 day postinfiltration and floated on deionized water overnight. Government (Generalitat de Catalunya). The authors declare no At 2 days postinfiltration, the leaf discs were treated with 100 nM conflicts of interest. flg22 or water for 1 hr and frozen for RNA extraction. Total RNA was extracted using TRI reagent (MRC) and treated with DNase I DATA AVA I L A B I L I T Y S TAT E M E N T (Sigma-Aldrich). RNA (2.5 μg) was used to synthesize cDNA using The data that support the findings of this study are available from a Maxima first-strand cDNA synthesis kit (Thermo Scientific). For the corresponding author upon reasonable request. quantitative PCR, cDNA was combined with GoTaq qPCR master mix (Promega) and PCR was performed in triplicate with a CFX con- ORCID nect real-time system (Bio-Rad). Expression was normalized to the Marc Valls https://orcid.org/0000-0003-2312-0091 reference gene NbEF1α. 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