Multiple effectors trigger nonhost resistance in Solanum americanum against Pseudomonas syringae

23 Wild plant species are threatened by diverse pathogens, but disease symptoms are rarely 24 observed in nature. This suggests that wild plants harbor valuable sources of resistance. In this 25 study, we show that a model bacterial pathogen Pseudomonas syringae pv. tomato (Pto) 26 DC3000 trigger ed defense responses in all tested accessions of a wild Solanaceae species, 27 Solanum americanum. Pto DC3000-triggered immunity in S. americanum required type III 28 secretion system . We show that seven Pto DC3000 effectors ( AvrPto, HopAD1, HopAM1, 29 HopC1, HopAA1 -1, HopM1, and AvrE1) trigger ed hypersensitive responses (HR) in S. 30 americanum accession SP2273 . Significantly, s equential deletion of the HR-triggering 31 effectors from Pto DC3000 resulted in enhanced virulence in S. americanum. However, the 32 well-conserved effectors, HopM1 and AvrE1 were indispensable for virulence. We conclude 33 that the immunity triggered by multiple effectors contributes to nonhost resistance in S. 34 americanum against P . syringae. We propose that the identification of the corresponding 35 disease resistance genes for HopM1 and AvrE1 in S. americanum would accelerate 36 development of durable immunity to P . syringae pathogens in Solanaceae crops. 37

Bacterial pathogenesis, nonhost resistance, effector, ETI, Solanum 38 americanum, Pseudomonas syringae 39 40

41 Plants are constantly threatened by the invasion of pathogens, yet plant diseases are relatively 42 uncommon in nature (Gill et al., 2015). This is due to the diverse defense strategies that plants 43 have evolved. There are two major immune layers (Jones and Dangl, 2006). The first layer is 44 pattern-triggered immunity (PTI), where pattern recognition receptors (PRRs) localized at the 45 plant cell surface detect conserved pathogen-associated molecules such as bacterial flagellin. 46 However, bacterial pathogens have developed sophist icated stra tegies to evade PTI by 47 delivering effector proteins to plant cells via type III secretion system (Macho, 2016). Many 48 bacterial effectors contribute to pathogen virulence by suppressing basal plant immunity. For 49 example, a well-studied Pseudomonas syringae effector, AvrPto interferes the kinase function 50 of certain PRRs such as Arabidopsis FLS2 or EFR, resulting in reduced PTI signaling (Xiang 51 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 3 et al., 2008; Zipfel and Rathjen, 2008) . In response to these effector activities, plants have 52 evolved a second layer of innate immunity termed effector-triggered immunity (ETI) where 53 nucleotide-binding and leucine-rich repeat resistance (NLR) proteins recognize corresponding 54 pathogen effectors (Jones and Dangl, 2006; Kourelis and van der Hoorn, 2018). ETI often leads 55 to hypersensitive response (HR) , characterized by localized cell death at the infection site. 56 Some NLR proteins, such as HOPZ-ACTIV ATED RESISTANCE1 (ZAR1), oligomerize upon 57 effector recognition and function as a calcium-permeable channel in the plasma membrane 58 which is required for HR development (Bi et al., 2021; Wang et al., 2019). 59 Pseudomonas sy ringae pv. tomato DC3000 (hereafter Pto DC3000) is a model 60 bacterial pathogen for studying plant-microbe interactions (Buell et al., 2003; Lindeberg et al., 61 2006; Xin and He, 2013) . In particular, the secretion and in planta functions of Pto DC3000 62 type III effectors have been extensively studied. Pto DC3000 secretes multiple effectors whose 63 functions can be complex and redundant. This nature makes it challenging to study individual 64 effector functions. To overcome these difficulties, combinations of multiple effectors were 65 deleted in Pto DC3000 background (Wei et al., 2007). Among 36 known Pto DC3000 type III 66 effectors, 28 are well-expressed (18 of these are clustered in six loci, while 10 effectors are 67 dispersed throughout the genome). The remaining eight effectors are either pseudogenes or 68 weakly expressed genes. Deletion of 18 clustered and well-expressed effector genes in Pto 69 DC3000 resulted in D18E, and deletion of all 28 well-expressed effector genes produced D28E 70 (Cunnac et al., 2011; Kvitko et al., 2009) . These Pto DC3000 polymutant strains showed 71 significantly reduced virulence in a model Solanaceae species Nicotiana benthamiana. Further 72 deletion of a weakly-expressed effector hopAD1 in D28E, resulted in D29E, which abolished 73 HR in N. benthamiana (Wei et al., 2015). Finally, the effectorless mutant D36E was generated 74 by deleting all the remaining weakly expressed effectors and pseudogenes from D29E (Wei et 75 al., 2015). Pto DC3000 effectors encoded by Exchangeable Effector Locus (EEL) vary among 76 different strains, whereas Conserved Effector Locus (CEL) encodes highly conserved effectors 77 such as AvrE1, HopM1, HopAA1 (HopN1 in some strains) a cross diverse P. syringae strains 78 (Alfano et al., 2000; Xin et al., 2018) . Since CEL effectors are highly conserved, identif ying 79 their corresponding resistance genes is considered to be crucial for developing durable disease 80 resistance to P . syringae pathogens (Dangl et al., 2013; Kim et al., 2022). 81 Wild plant species are valuable sources of resistance genes compared to domesticated 82 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 4 crops (Arora et al., 2019) . Several P . syringae effectors have been shown to activate NLR -83 mediated immunity in N. benthamiana, a model Solanaceae species. HopQ1 effector from P . 84 syringae, for example, is recognized by NLR protein Roq1 with N-terminal Toll-like 85 interleukin-1 (TIR) domain (Schultink et al., 2017) . Interestingly, Roq1 also recognizes 86 Xanthamonas and Ralstonia effectors, XopQ and RipB, respectively. Recently, N. benthamiana 87 and Solanum lycopersicoides NLR Ptr1, which contains coiled-coil (CC) domain, was shown 88 to recognize multiple bacterial effectors. For instance, P . syringae effectors AvrRpt2, AvrRpm1, 89 AvrB, and HopZ5 activate NbPtr1-dependent immunity in N. benthamiana (Ahn et al., 2023). 90 Moreover, NbPtr1 also recognize s RipBN and RipE1 from Ralstonia sola nacearum, and 91 AvrBsT from Xanthomonas euvesicatoria. Solanum americanum, a wild Solanaceae species, 92 has been used to identify NLR genes (Witek et al., 2016). High-quality genome assemblies and 93 NLR gene repertoires have been analyzed in multiple S. americanum accessions in recent 94 research (Lin et al., 2023; Witek et al., 2016). Moreover, the availability of genetically variable 95 S. americanum accessions makes S. americanum an ideal model for discovering NLR genes 96 (Witek et al., 2016). Several NLR genes that recognize effectors from Phytophthora infestans 97 causing potato late blight have been identified and cloned from S. americanum (Lin et al., 2023; 98 Witek et al., 2016; Witek et al., 2021). For instance, Rpi-amr1 from S. americanum recognizes 99 Avr-amr1 and provides resistance when expressed in other Solanaceae species, such as potato 100 and N. benthamiana. Additionally, Rpi-amr3 recognizing Avr-amr3 confers broad resistance to 101 P . infestans and other Phytophthora pathogens including P . parasitica and P . palmivora when 102 expressed in N. benthamiana (Lin et al., 2022). 103 In this study, we aimed to understand the genetic basis of S. americanum resistance to 104 Pto DC3000 and found that seven effectors (AvrPto, HopAD1, HopAM1, HopC1, HopAA1-1, 105 HopM1, and AvrE1) trigger HR in S. americanum accession SP2273. We generated Pto 106 DC3000 mutants lacking these HR-triggering effectors and investigated their roles in bacterial 107 disease resistance in S. americanum. We show that the deletion of avrPto, hopAD1, hopAM1, 108 hopC1, and hopAA1-1 from Pto DC3000 enhance s in planta bacterial growth and causes 109 bacterial speck symptoms while still inducing weak HR in S. americanum. Deletion of all seven 110 avirulence effectors abolished the HR -triggering ability of Pto DC3000 in S. americanum 111 without disease development . Based on these results, we propose that multiple effectors are 112 required for nonhost resistance in S. americanum against P . syringae. Our results offer insights 113 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 5 into bacterial virulence mechanisms and can help the identification of NLRs recognizing CEL 114 effectors, which may confer durable resistance in Solanaceae crops. 115 116

117 Pseudomonas syringae pv. tomato DC3000 triggers type III secretion system -dependent 118 disease resistance in Solanum americanum 119 To better understand the genetic basis of disease resistance to Pseudomonas syringae in S. 120 americanum, we infiltrated Pseudomonas syringae pv. tomato (Pto) DC3000 (OD 600nm=0.1) 121 into the leaves of 28 S. americanum accessions and tested for the onset of hypersensitive 122 response (HR) . Unlike P . infestans and R. pseudosolanacearum , which induce accession -123 specific resistance in S. americanum (Moon et al., 2021; Witek et al., 2016), Pto DC3000 wild-124 type strain triggered a strong HR in all tested S. americanum accessions at one day post -125 infection (dpi) (Table 1). We hypothesized that one or more Pto DC3000 type III effector (T3E) 126 proteins trigger HR in S. americanum . In order t o identify which Pto DC3000 T3E(s) are 127 responsible for HR, we tested HR-inducing activity of Pto DC3000 polymutants D18E, D29E, 128 and D36E lacking multiple T3Es (Kvitko et al. , 2009; Wei et al. , 2015) in S. americanum 129 accession SP2273 (hereafter, SP2273). Pto DC3000 wild-type and D18E triggered strong HR, 130 whereas D29E and D36E did not induce any visible symptoms in SP2273 (Figure 1A). Next, 131 we tested the virulence of Pto DC3000 wild-type and polymutants by measuring in planta 132 bacterial growth. None of the strains (Pto DC3000 wild-type, D18E, D29E, or D36E) showed 133 growth in SP2273 (Figure 1B), suggesting that deletion of multiple T3Es resulted in the loss of 134 not only HR but also bacterial virulence in S. americanum . Based on these results, we 135 hypothesized that the 11 effectors present in D18E but absent in D29E (HopA1, HopAD1, 136 HopAF1, HopAM1, Ho pB1, HopE1, AvrPto, AvrPtoB, HopI1, HopK1, and HopY1) are 137 primary avirulence effector candidates (Figure 1C). In addition, the 18 effectors that are present 138 in wild -type strain but absent in D18E (HopAA1-1, HopAA1-2, HopAO1, AvrE1, HopC1, 139 HopD1, HopR1, Hop G1, HopH1, HopM1, HopN1, HopO1 -1, HopQ1 -1, HopF2, HopT1 -1, 140 HopU1, HopV1, and HopX1) could be the additional avirulence effector candidates (Fig 1C). 141 We excluded the seven effectors (HopS2, HopO1 -3’, HopO1-2, HopS1’, HopBM1, HopT2, 142 and HopT1-2’) remaining in D29E because D29E failed to trigger HR, and these genes are 143 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 6 considered weakly expressed genes or pseudogenes (Wei et al., 2015). Taken together, these 144

strongly suggest that T3Es are essential for Pto DC3000-induced HR in S. americanum. 145 146 AvrPto, HopAM1, or HopAD1 triggers hypersensitive response in Solanum americanum 147 To identify avirulence effector(s) that trigger defense responses in S. americanum , we 148 transiently expressed each of the 11 effectors present in D18E (HopA1, HopAD1, HopAF1, 149 HopAM1, HopB1, HopE1, AvrPto, AvrPtoB, HopI1, HopK1, and HopY1) in SP2273 leaves 150 by Agrobacterium-mediated transient transformation (hereafter, agroinfiltration). Among these, 151 agroinfiltration of HopAD1, HopAM1, or AvrPto elicited a robust HR in SP2273 (Figure 2A). 152 To test whether the lack of HR for other effectors was due to protein instability. We transiently 153 expressed all 11 effectors in Nicotiana benthamiana leaves via agroinfiltration and total protein 154 extracts were analyzed by immunoblot using anti-HA antibody. All tested effectors showed 155 detectable protein expression levels (Figure 2B and Table S5). Although HopAM1 showed 156 weaker expression compared to other effectors, it still induced HR (Figure 2A), indicating that 157 the level of expression was sufficient to activate plant defense responses. 158 159 Pto DC3000 lacking avrPto, hopAM1, and hopAD1 shows enhanced in planta bacterial 160 growth, yet still triggers hypersensitive response in Solanum americanum 161 To investigate the role s of AvrPto, HopAM1, and HopAD1 in Pto DC3000 virulence, we 162 sequentially deleted each effector gene and tested for HR induction and in planta bacterial 163 growth in S. amerianum. Effector knock-out mutants were generated using a modified suicide 164 vector pK18mobsacB-GG containing the upstream and downstream flanking regions of the 165 target effector genes (Jayaraman et al., 2020). First, we deleted avrPto from Pto DC3000 wild-166 type resulting in strain PKSG 4673 (Figure 3A). Since hopAM1 gene exists in two identical 167 copies, one on the chromosome ( hopAM1-1) and the other on the plasmid , pDC3000A 168 (hopAM1-2) (Buell et al., 2003), we deleted hopAM1-1 from PKSG 4673 (PKSG 7065) and 169 subsequently deleted hopAM1-2 from PKSG 7065 (PKSG 7903). Finally, we deleted hopAD1 170 from PKSG 7903 , generating PKSG 7377. Details on the generation and validation of these 171 effector knockout strains are provided in the Methods section and Figure S1. 172 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 7 To test whether these effector knockout mutants exhibited enhanced virulence , we 173 measured in planta bacterial growth by infiltrating a low concentration of bacterial suspension 174 (OD600nm=0.0001) into SP2273 leaves using a needleless syringe . All mutant strains, PKSG 175 4673 (ΔavrPto), PKSG 7065 (ΔavrPto hopAM1-1), PKSG 7903 (ΔavrPto hopAM1-1 hopAM1-176 2), PKSG 7377 (ΔavrPto hopAM1-1 hopAM1-2 hopAD1) showed significantly higher in planta 177 growth compared to wild-type (Figure 3B). However, no significant differences were observed 178 among the mutant strains. To further characterize these mutant strains, we tested their HR-179 inducing ability by infiltrating a high concentration of bacterial inoculum (OD600nm=0.1) into 180 SP2273 leaves. Interestingly, all four Pto DC3000 mutant strains still triggered a strong HR in 181 SP2273 (Figure 3C and 3D). Taken together, these results indicate that AvrPto, HopAM1, and 182 HopAD1 significantly contribute to the avirulence of Pto DC3000 in S. americanum. However, 183 the remaining HR suggests that additional avirulence determinant(s) are likely present in Pto 184 DC3000. 185 186 Transient expression of HopC1, HopAA1-1, HopM1 or AvrE1 triggers hypersensitive 187 response in Solanum americanum 188 To identify additional avirulence effector(s), we tested the HR-inducing activity of 18 189 secondary candidates (HopAA1-1, HopAA1 -2, HopAO1, AvrE1, HopC1, HopD1, HopR1, 190 HopG1, HopH1, HopM1, HopN1, HopO1 -1, HopQ1 -1, HopF2, HopT1 -1, HopU1, HopV1, 191 and HopX1) in SP2273 (Figure 1C). Agroinfiltration of these effectors in to SP2273 leaves 192 showed that HopAA1-1, AvrE1, HopC1, or HopM1 induced a strong HR at 3 dpi (Figure 4A). 193 HopAA1-1, AvrE1, and HopM1 are known to be highly conserved effectors among diverse P. 194 syringae strains (Alfano et al. , 2000; Munkvold et al. , 2009) . We n ext assessed protein 195 expression of these effectors by immunoblot analysis using an anti-HA antibody. Most effectors 196 showed detectable levels of expression . However, for unknown reasons, t he expected size 197 bands for AvrE1 (202 kDa) and HopR1 (217 kDa) (Table S5) were not detected in our 198 experimental conditions (Figure 4B). 199 200 HopM1 and AvrE1 are critical for bacterial virulence and disease symptom development 201 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 8 in Solanum americanum 202 To test the roles of HopC1, HopAA1-1, HopM1, and AvrE1 in HR induction and bacterial 203 virulence, we generated additional effector knockout mutant strains of Pto DC3000. Details for 204 generation and validation of these effector knockout strains can be found in the Methods section 205 and Figure S3. HopM1 and AvrE1 require chaperone proteins ShcM and ShcE, respectively, 206 for proper functions (Badel et al., 2003; Badel et al., 2006) . Therefore, shcM and shcE were 207 deleted along with their corresponding effector genes, hopM1 and avrE1. First, hopC1 was 208 deleted in PKSG 7377 which resulted in ΔavrPto hopAM1 -1 hopAM1 -2 hopAD1 hopC1 209 (PKSG 7768) (Figure 5A). Next, hopAA1-1 was deleted in PKSG 7768 to generate PKSG 7826. 210 We further deleted shcM-hopM1 or/and schE -avrE1 resulting in three additional knockout 211 strains (PKSG 7899 ; shcM-hopM1 deletion, PKSG 7900 ; shcE-avrE1 deletion, and PKSG 212 7892; both deletions) (Figure 5A). 213 To investigate the effect of these additional effector deletions on virulence, we 214 conducted in planta bacterial growth assays in SP2273. Infection conditions were identical as 215 described in Figure 3 B. Similar to PKSG 7377, PKSG 7768, PKSG 7826, and PKSG 7899 216 mutants showed significant increase in growth compared to Pto DC3000 wild-type (Figure 5B). 217 However, in planta growth of PKSG 7900 and PKSG 7892 lacking shcE-avrE1 was not 218 significantly different from Pto DC3000 wild -type (Figure 5B). Next, we tested the HR-219 inducing activity of these mutant strains in SP2273. Interestingly, PKSG 7768, PKSG 782 6, 220 PKSG 7899, and PKSG 7900 triggered significantly reduced HR compared to Pto DC3000 221 wild-type or PKSG 7377 (Figure 5C and 5D). Notably, HR was completely abolished in PKSG 222 7892, which lacks all effectors previously shown to induce HR in agroinfiltration assay. Finally, 223 we conducted an additional infection assay to monitor disease symptom s caused by effector 224 knockout mutant strains. When SP2273 leaves were sy ringe-infiltrated with a low 225 concentration of bacterial inoculum (OD 600nm=0.00001), PKSG 7826 caused visible bacterial 226 speck symptoms (Figure 6A). PKSG 7899 caused a weaker, yet still notable symptoms, while 227 other strains did not produce visible disease symptoms. We further confirmed this phenotype 228 by dip-inoculation assays. We dip-inoculated SP2273 plants with PKSG 7826 (OD600nm=0.001). 229 PKSG 7826 showed a mild bacterial speck disease symptom, while Pto DC3000 wild-type did 230 not cause visible disease symptoms under the same conditions (Figure 6B). In summary, these 231

demonstrate that HopC1, HopAA1-1, HopM1, and AvrE1 are required for Pto DC3000 232 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 9 avirulence in S. americanum. Moreover, despite their HR-inducing activity, HopM1 and AvrE1 233 appear to carry strong virulence functions as deletion of these effectors significantly reduced 234 in planta bacterial growth. 235 Conservation of avirulence effectors across multiple Pseudomonas syringae strains. 236 Identification of the NLR genes that recognize broadly conserved effectors is considered to 237 provide a source of durable disease resistance. To survey the conservation of seven avirulence 238 effectors identified in this study , we analyzed their presence/absence polymorphism in 117 239 phytopathogenic Pseudomonas strains with available genome sequences in the NCBI database, 240 using effector references from the PsyTEC library (Laflamme et al., 2020) . The number of 241 Pseudomonas species or pathovar strains analyzed in this research is shown in Table S10. 242 Proteins with an E-value above 1e-24 or those with less than 60 % sequence coverage as 243 compared to the effector alleles in PsyTEC database were considered absent (Figure 7A). Based 244 on this criterion, HopAD1, HopAM1, AvrPto, HopC1, HopAA1, HopM1 , and AvrE1 were 245 present in 8 (6.8%), 25 (21.4%), 34 (29.1%), 14 (12%), 70 (59.8%), 75 (64.1%) and 109 (93.2%) 246 out of 117 strains, respectively (Figure 7B). 247 248

249 Plant immunity triggered by multiple effectors is involved in nonhost resistance. 250 To better understand t he interaction between Solanum americanum and pathogenic 251 Pseudomonas syringae strains, we focused on the well-characterized strain, Pto DC3000. 252 Unlike other pathogens such as P . infestans and R. pseudosolanacearum (Moon et al., 2021; 253 Witek et al. , 2016) , Pto DC3000 induced defense responses in all tested S. americanum 254 accessions (Table 1). Furthermore, well-conserved P . syringae effectors such as HopAA1-1, 255 HopM1, and AvrE 1 activated defense responses in S. americanum , suggesting that S. 256 americanum may be a nonhost plant species to P . syringae. 257 The molecular basis of nonhost resistance is still not fully understood, and it is thought 258 to involve multiple contributing factors (Panstruga and Moscou, 2020) . One proposed 259 mechanisms is the recognition of pathogen effectors by corresponding NLR genes. For instance, 260 Phytophthora sojae is typically a non-adapted pathogen to N. benthamiana. However, deletion 261 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 10 of AvrNb, an effector recognized by the immune receptor NbPrf, enables P . sojae to infect N. 262 benthamiana (Dong et al., 2025) . This highlights the role of ETI in nonhost resistance. 263 Consistent with our results, previous studies have shown that nonhost resistance can be 264 mediated by the recognition of multiple pathogen effectors. For example, Cevik et al. identified 265 Albugo candida -susceptible transgressive segregated lines by using Arabidopsis thaliana 266 multiparent advanced generation intercross (MAGIC) lines. Their findings highlighted that 267 nonhost resistance in A. thaliana is polygenic and induced through the recognition of multiple 268 Albugo candida effectors (Cevik et al., 2019). Similarly, studies in pepper (Capsicum annuum) 269 also support th is concept. Lee et al. identified several RxLR effectors from P . infestans that 270 triggered HR in diverse pepper accessions. It suggests that recogni tion of multiple effectors 271 contributes to nonhost resistance (Lee et al., 2014). Also, Oh et al., showed that stack ed NLR 272 genes in pepper mediate nonhost resistance by recognizing distinct effectors (Oh et al., 2023). 273 While these studies demonstrate nonhost resistance from the plant’s perspective, our research 274 provides additional evidence from the pathogen’s perspective. Specifically, we show that the 275 deletion of multiple HR-triggering effectors enables previously nonpathogenic strain to cause 276 disease in S. americanum. Therefore, our research supports the idea that multiple ETIs is one 277 of the genetic bases of nonhost resistance. 278 279 HopM1 and AvrE1 are highly conserved among Pseudomonas strains and critical for 280 virulence in Solanum americanum. 281 In this study, we show that HopM1 and AvrE1 are crucial for the full virulence of Pto DC3000 282 in S. americanum. Previously, multiple studies showed the importance of HopM1 and AvrE1 283 in pathogen virulence. For instance, deletion of hopM1 and avrE1 in Pto DC3000 reduces 284 growth and lesion formation in tomato (Badel et al. , 2006) . In P . syringae pv. actinidae, 285 although HopM1 is non-functional due to the loss of function mutation in schM, AvrE1 286 significantly contributes to virulence in kiwifruit (Jayaraman et al. , 2020) . Furthermore, 287 DspA/E and WtsE, belonging to the AvrE family, are crucial for the full virulence of Erwinia 288 amylovora and Pantoea stewartii, respectively (Degrave et al., 2015) . More recently, AvrE1 289 and HopM1 were shown to be critical for bacterial virulence in spinach (Mendel et al., 2024). 290 Interestingly, HopM1 and AvrE1 were shown to be critical for the development of water-291 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 11 soaking symptoms during bacterial infection (Xin et al. , 2016). HopM1 and AvrE1 induce 292 stomatal closure by activating abscisic acid (ABA) signaling , generating an aqueous 293 environment in the apoplast that is favorable for bacterial proliferation (Roussin-Leveillee et 294 al., 2022). Consistent with these findings, our results support the virulence function of HopM1 295 and AvrE1 in S. americanum. It is conceivable that other effectors in Pto DC3000 suppress the 296 avirulence activity of HopM1 and AvrE1 in S. americanum . It was shown that immune 297 responses mediated by conserved effectors such as HopAA1, HopM1, and AvrE1 can be 298 suppressed by the function of other effectors (Wei et al., 2007). For example, HopI1 suppresses 299 cell death triggered by AvrE1, HopM1, HopQ1-1, HopR1, or HopAM1 in N. benthamiana (Wei 300 et al., 2018). Thus, this suggests that these HopM1 and AvrE1 might be indispensable for the 301 full virulence of Pto DC3000, although they have the disadvantage of being recognized by 302 unknown plant immune receptors in S. americanum . While HopM1 and AvrE1 have been 303 described to have functional redundancy (Kvitko et al., 2009), our results show that deletion 304 of either effector leads to reduced bacterial growth in S. americanum. This discrepancy may 305 reflect differences in the host resistance gene repertoire or other deleted effectors could 306 influence different levels of redundancy. 307 308 Identifying NLR genes that recognize HR-triggering effectors may enable us to develop 309 durable bacterial speck disease resistant Solanaceae crops. 310 Nonhost resistance confers broad and durable resistance against pathogens (Fonseca and 311 Mysore, 2019). In this study, we hypothesized that nonhost resistance in S. americanum is 312 mediated by multiple ETIs. Therefore, identifying NLR genes that recognize effectors involved 313 in nonhost resistance may provide tools for developing durable P. syringae-resistance in 314 Solanaceae crops. Several a virulence effectors identified in this study also trigger immune 315 responses in other plant species. For example, HopAD1 induces immune-associated cell death 316 in N. benthamiana (Wei et al., 2015). AvrPto is known to bind to Pto kinase, activating Prf-317 dependent disease resistance in tomato . We identif ied Pto and Prf homologs in the S. 318 americanum SP2273 genome , suggesting that recognition of AvrPto in this accession may 319 occur via a similar mechanism with tomato. Furthermore, HopAM1 from P . syringae pv. 320 actinidae which shares 98.9 % amino acid identity with Pto DC3000 allele also induces cell 321 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 12 death in Nicotiana species, and the homolog from P . syringae pv. pisi induces cell death in pea 322 cultivars (Choi et al., 2017; Cournoyer et al., 1995; Eastman et al., 2022). HopAM1 contains a 323 Toll/interleukin-1 receptor (TIR) domain such as TIR-type NLR , which activates immune 324 signaling and cell death in plants (Eastman et al., 2022). Therefore, HopAM1 may trigger cell 325 death independently of a specific resistance gene. HopC1 homolog from P . syringae pv. pisi 326 which shares 99.6 % amino acid identity with Pto DC3000 allele, acts as an avirulence effector 327 in bean (Arnold et al., 2001; Baltrus et al., 2012). HopM1 and AvrE1 also trigger cell death in 328 N. benthamiana (Wei et al., 2018). Despite various research on HR phenotypes and immune 329 responses for theses effectors, resistance genes recognizing these avirulence effectors are not 330 well-studied in Solanaceae plants. In Arabidopsis, the immune receptor, CEL-ACTIV ATED 331 RESISTANCE1 (CAR1), recognizes AvrE1 and HopAA1-1 (Laflamme et al., 2020). However, 332 an NCBI BLAST search using the CAR1 sequence (AT1G50180.1) found no clear homolog in 333 the SP2273 genome (data not shown). This could be an example of convergent evolution, where 334 an effector protein is recognized by resistance genes with no sequence similarity in different 335 plant species (Kim et al., 2023). In the future, NLR genes recognizing avirulence Pto DC3000 336 effectors could be identified through natural variations of S. ameri canum accessions. In 337 particular, NLRs that detect conserved avirulence effectors such as AvrE1, HopM1, and 338 HopAA1 may be especially valuable for developing durable resistance against P . syringae in 339 Solanaceae crops. 340 341 Virulent Pto DC3000 mutant strain is a valuable tool for studying effector -triggered 342 immunity in Solanum americanum. 343 S. americanum is a promising model Solanaceae plant for identifying resistance genes against 344 diverse phytopathogens (Witek et al. , 2016) . However, the lack of virulent strains for S. 345 americanum makes it challenging to study the interaction between effectors and host plants. In 346 this study, we generated virulent strains using the model bacterial pathogen Pto DC3000. In N. 347 benthamiana, a well-established model Solanaceae plant, Pto DC3000 ∆hopQ1-1 is commonly 348 used for in planta bacterial growth assays (Wei et al., 2007). Similarly, we can use the virulent 349 PKSG 7826 strain to perform in planta bacterial growth assays in S. americanum. Thus, this 350 study highlights the potential of S. americanum to serve as a model Solanaceae plant for ETI 351 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 13 studies alongside N. benthamiana. 352 353

354 Plant growth conditions 355 Solanum americanum and Nicotiana benthamiana plants were grown in Baroker soil mix (4 % 356 zeolite, 7 % perlite, 6 % vermiculite , 68 % cocopeat, 14.73 % peat moss; Seoulbio , 357 http://www.seoulbio.co.kr) at 23 ℃ with 11 hours of light per day. S. americanum was used for 358 agroinfiltration and Pseudomonas syringae pv. tomato DC3000 in planta growth assays. N. 359 benthamiana was used for agroinfiltration followed by total protein extraction and immunoblot 360 analysis. 361 362 Bacterial strains and culture conditions 363 The bacterial strains used in this study are listed in Table 2. Escherichia coli DH5α and 364 Agrobacterium tumefaciens AGL1 strains were cultured in Luria-Bertani (LB) broth containing 365 appropriate antibiotics. Pseudomonas syringae strains were grown on King’s B (KB) medium 366 with appropriate antibiotics. Antibiotics for bacterial strains are shown in Table S2. Antibiotic 367 concentrations used were: Carbenicillin (100 μg/mL), Kanamycin (50 μg/mL), Gentamycin (20 368 μg/mL), Rifampicin (50 μg/mL), Spectinomycin (100 μg/mL). E. coli was grown at 37 ℃, and 369 A. tumefaciens and P . syringae were grown at 28 ℃. 370 371 Construction of plasmids 372 To clone Pto DC3000 type III effector gene s, we used Pto DC3000 chromosome data 373 (GenBank: AE016853.1) and pDC3000a plasmid data (NCBI: NC_004633.1). If effector 374 sequences are over 2000 bp, we divided them into modules (about 500 - 1500 bp) for efficient 375 golden-gate (GG) assembly. First, BsaI site flanked nucleotide sequences of hopA1, hopAD1, 376 hopAF1 (BsaI mutagenized), hopAM1, hopB1 (BsaI mutagenized), hopE1, hopI1 (codon 377 optimized), hopK1, hopY1, hopD1_module2 ( BsaI mutagenized), hopR1_module6 (BsaI 378 mutagenized), hopN1 (BsaI mutagenized), hopAA1-2_module2 ( BsaI mutagenized), 379 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 14 avrE1_module 4 (BsaI mutagenized) were synthesized ( Twist Biosciences, South San 380 Francisco, CA, USA). Other effectors or effector modules were PCR amplified. Pto DC3000 381 genomic DNA (extracted using Wizard Genomic DNA purification kit; Promega , 382 https://www.promega.com/) was used as the PCR template. All DNA fragments were flanked 383 by BsaI restriction enzyme site and GG overhangs to make GG modules. Effectors were cloned 384 into pICH41021 vector (BsaI mutagenized pUC19, hereafter pUC19B) by blunt-end ligation 385 using SmaI or Eco53KI restriction enzyme. The pUC19B modules were cloned into the binary 386 vector (pICH86988) with a C -terminal 6x HA epitope tag using golden gate assembly using 387 BsaI restriction enzyme (Engler et al., 2008). 388 For generating Pto DC3000 type III effector knockout plasmi ds, the upstream and 389 downstream of the target effector region (around 1~1.8 kb) were PCR -amplified. DNA 390 fragments were flanked by BsaI restriction site s and GG overhangs. The amplified PCR 391 templates were cloned into pUC19B vector. The upstream and downstream pUC19B modules 392 were assembled into suicide pK18mobsacB-GG vector via GG assembly using BsaI restriction 393 enzyme (Jayaraman et al. , 2020) . pK18mobsacB-GG vector was derived by removing the 394 multiple cloning site (MCS) of the original pk18mobsacB vector. The removed MCS was 395 replaced with a BsaI restriction site flanked MCS of pICH86988 (Jayaraman et al., 2020). The 396 primers or synthesized sequences for pUC19B module cloning are listed in Table S11 and Table 397 S12. All pUC19B modules and GG-assembled constructs were transformed into E. coli DH5α 398 using electroporation method (1.8 kV pulse using 1 mm electroporation cuvette) . E. coli 399 transformants were selected on LB media containing appropriate antibiotics for destination 400 vectors (see Table S3 for details). The insert sequences of pUC19B modules w ere validated 401 using Sanger sequencing and GG-assembled constructs were verified using restriction enzyme 402 digestion. 403 404 Agrobacterium-mediated transient transformation 405 All effectors used in this study were cloned in the binary vector pICH86988 for Agrobacterium-406 mediated transient transformation (agroinfiltration). These binary constructs were transformed 407 into A. tumefaciens AGL1 strain using electroporation (2.2 kV pulse using 1mm electroporation 408 cuvette). AGL1 transformants were selected on LB media containing Carbenicillin (100 μg/mL) 409 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 15 and Kanamycin (50 μg/mL). For agroinfiltration, AGL1 strains carrying effector constructs and 410 P19, a viral suppressor of RNA silencing, were grown in liquid LB with appropriate antibiotics. 411 The cultured cells were resuspended in Agrobacterium infiltration buffer (10 mM MgCl 2 and 412 10 mM MES (pH 5.6) ) and diluted to OD 600nm 0.2 (P19) to 0.4 (effector). The diluted 413 Agrobacterium inoculums carrying effector constructs and P19 were mixed and infiltrated 414 using a 1 mL needleless syringe into plant leaves. 415 416 Immunoblot analysis 417 The total protein was extracted from six leaf discs (8mm diameter) taken from the infiltrated 418 area of N. benthamiana two days post infiltration. The leaf discs were snap -frozen in liquid 419 nitrogen and ground with 200 μL of 5X SDS protein loading buffer (250mM Tris-HCl (pH 6.8), 420 8 % sodium dodecyl sulfate (SDS), 0.1 % Bromophenol blue, 40 % (v/v) Glycerol , and 100 421 mM Dithiothreitol). The total protein samples were boiled for 10 minutes at 95 ℃. For SDS 422 page, 20 μL of protein samples were loaded in polyacrylamide gel and ran for 1 hour (130 V). 423 HopA1, HopAD1, HopAF1, HopAM1, HopB1, HopE1, AvrPto, AvrPtoB, HopI1, HopK1, 424 HopY1, HopH1, HopM1, HopN1, HopO1 -1, HopQ1 -1, HopF2, HopT1 -1, HopU1, HopV1, 425 and HopX1 protein samples were loaded in 10 % polyacrylamide gel. HopAA1-1, HopAA1-2, 426 HopAO1, AvrE1, HopC1, HopD1, HopR1, and HopG1 were loaded in 4~12 % Mini-427 PROTEAN TGX Precast Gels (Bio-Rad, https://www.bio-rad.com/). Proteins were transferred 428 from the polyacrylamide gel into PVDF membranes for one hour (100 V). Protein-transferred 429 PVDF membranes were blocked using 5 % (w/v) skim milk in Tris-Buffered Saline (pH 7.4), 430 and 0.1 % Tween20 (TBST) for 30 minutes. After blocking, anti -HA (Roche 11867423001; 431 1:2000 dilution) antibodies were added to the blocking buffer and incubated for 1 hour at room 432 temperature. The membrane was washed for five minutes ( five times ) using TBST. The 433 membranes were blocked in a blocking buffer for 30 minutes at room temperature . Anti-rat 434 secondary antibody (Sigma A9037; 1:20000 dilution) was added to the blocking buffer. The 435 membranes were incubated for 1 hour 30 minutes at room temperature. Membranes were 436 washed for five minutes (five times) using TBST. Proteins were visualized using Super Signal 437 West Pico and Femto Chemiluminescent substrate (Thermo Fisher Scientific, 438 https://www.thermofisher.com/) through ChemiDoc XRS+ with Image Lab Software (Bio -439 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 16 Rad). After visualization, Membranes were stained with Ponceau S to estimate the quantity of 440 proteins. 441 442 Hypersensitive response, in planta growth, and dip inoculation assays using Pseudomonas 443 Pseudomonas syringae pv. tomato DC3000 wild-type and mutant strains were grown on KB 444 agar with appropriate antibiotics. Pto DC3000 cells were resuspended in autoclaved 445 Pseudomonas infiltration buffer (10 mM MgCl 2). The inoculum was diluted to OD 600nm=0.1 446 for HR assays , OD600nm=0.0001 for in planta growth assay s and OD600nm=0.001 for dip 447 inoculation. The bacterial inoculum was infiltrated using a needleless syringe into four to five-448 week-old S. americanum leaves. HR was scored from 0 to 7 one day post infiltration. HR 449 scoring criteria were described in Figure S2 and revised from the previous study (Ahn et al., 450 2023). The HR scores triggered by Pto DC3000 wild-type and effector knockout mutant s are 451 shown in the violin plots showing individual replicate data. For in planta bacterial growth, two 452 leaf discs (8 mm diameter) were grounded and diluted in 500 μL of autoclaved Pseudomonas 453 infiltration buffer. Serial dilutions (10 μL) were spotted on KB agar with appropriate antibiotics. 454 Colonies were counted after two days at 28 ℃. Three to four plants were used per batch repeat. 455 The bacterial growth (CFU/cm 2) is shown in the bar graph with individual data. For dip 456 inoculation, the bacteria inoculum was diluted to OD600nm=0.001 in Pseudomonas infiltration 457 buffer and 0.05 % Silwet L-77. Four- to five-week-old SP2273 leaves were dipped and gently 458 swirled for 2 minutes. The dipped S. americanum leaves were covered for one day to maintain 459 humidity (11h light, 23 ℃). The photographs were taken 12 days after dip inoculation. 460 461 Generation of Pseudomonas syringae pv. tomato DC3000 effector knock-out strains 462 Details of effector deletion in Pto DC3000 can be found in (Jayaraman et al., 2020). Recipient 463 bacteria were incubated on KB agar with Rifampicin ( 50 μg/mL). Helper E. coli HB101 and 464 Donor E. coli DH5α carrying suicide vector (pK18mobsacB -GG) containing upstream and 465 downstream regions of the target effector were cultured on LB medium containing Kanamycin 466 (50 μg/mL). All three bacteria strains were mixed on LB agar without antibiotics and incubated 467 for 6-7 hours at 28 ℃. Mixed bacteria were streaked on KB agar with Rifampicin (50 μg/mL) 468 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 17 and Kanamycin (50 μg/mL). After 2 days of incubation at 28 ℃, a single colony was selected. 469 A single colony was inoculated into liquid LB media with Rifampicin (50 μg/mL) and grown 470 for one day at 28 ℃. The cultured bacteria were streaked on KB agar media containing 471 Rifampicin (50 μg/mL) and 10 % sucrose (w/v). After two days of incubation at 28 ℃, a single 472 colony was selected. Successful knockouts showed reduced band size s using specific PCR 473 primers (see Table S11 for details). 474 475 DATA A V AILABILITY 476 All relevant data can be found in the manuscript , Supplemental information, and public 477 databases (NCBI). 478 479 FUNDING 480 This research was supported by the National Research Foundation of Korea (NRF) grants 481 funded by the Korean government (MIST) (RS-2025-00512558 and 2023R1A2C3002366) and 482 Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry 483 (IPET) through the Agriculture and Food Convergence Technologies Program for Research 484 Manpower Development, funded by the Ministry of Agriculture, Food and Rural Affairs 485 (MAFRA) (No. RS-2024-00398300). 486 487 AUTHOR CONTRIBUTIONS 488 JK and KHS designed the experiments. JK performed the experiments. JK, MVZ, and KHS 489 analyzed the data. JK and KHS wrote the manuscript. 490 491

492 We thank Prof. Duck Hwan Park ( Kangwon National University, Republic of Korea) and Dr. 493 Jay Jayaraman (Plant and Food Research, New Zealand) for sharing materials. The authors 494 declare no competing interests. 495 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 18 496 SUPPLEMENTAL INFORMATION 497 Figures S1. Generation and confirmation of primary candidate effectors knockout, related to 498 Figure 3 499 Figure S2. Hypersensitive response scoring criteria in Solanum americanum, Related to 500 Figure 3 and Figure 5 501 Figure S3. Generation and confirmation of effector knockout, Related to Figure 5 502 Table S1. Solanum americanum accessions used in this study 503 Table S2. Bacterial strains used in this study 504 Table S3. Plasmids used in this study 505 Table S4. Raw data of in planta bacterial growth, Related to Figure 1 506 Table S5. Type III effector expected protein size, Related to Figure 2 and Figure 4 507 Table S6. Raw data of in planta bacterial growth, Related to Figure 3 508 Table S7. Raw data of hypersensitive response scoring, Related to Figure 3 509 Table S8. Raw data of in planta bacterial growth, Related to Figure 5 510 Table S9. Raw data of hypersensitive response scoring, Related to Figure 5 511 Table S10. Numbers of species or pathovars analyzed in this study, Related to Figure 7 512 Table S11. Excel file containing information of primers used in this study 513 Table S12. Excel file containing sequence information of synthesized constructs 514 515 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 19

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Modular study of the type III effector 670 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 24 repertoire in Pseudomonas syringae pv. tomato DC3000 reveals a matrix of effector interplay 671 in pathogenesis. Cell reports 23:1630-1638. 672 Wei, H. -L., Chakravarthy, S., Mathieu , J., Helmann, T.C., Stodghill, P., Swingle, B., 673 Martin, G.B., and Collmer, A. (2015). Pseudomonas syringae pv. tomato DC3000 type III 674 secretion effector polymutants reveal an interplay between HopAD1 and AvrPtoB. Cell host & 675 microbe 17:752-762. 676 Witek, K., Jupe, F., Witek, A.I., Baker, D., Clark, M.D., and Jones, J.D. (2016). Accelerated 677 cloning of a potato late blight -resistance gene using RenSeq and SMRT sequencing. Nat 678 Biotechnol 34:656-660. 10.1038/nbt.3540. 679 Witek, K., Lin, X., Karki, H., Jupe, F., Wite k, A., and Steuernagel, B. (2021). A complex 680 resistance locus in Solanum americanum recognizes a conserved Phytophthora effector. Nat 681 Plants. 2021; 7: 198–208. 682 Xiang, T., Zong, N., Zou, Y., Wu, Y., Zhang, J., Xing, W., Li, Y., Tang, X., Zhu, L., Chai, 683 J., and Zhou, J.M. (2008). Pseudomonas syringae effector AvrPto blocks innate immunity by 684 targeting receptor kinases. Curr Biol 18:74-80. 10.1016/j.cub.2007.12.020. 685 Xin, X.-F., and He, S.Y. (2013). Pseudomonas syringae pv. tomato DC3000: a model pathogen 686 for p robing disease susceptibility and hormone signaling in plants. Annual review of 687 phytopathology 51:473-498. 688 Xin, X.F., Kvitko, B., and He, S.Y. (2018). Pseudomonas syringae: what it takes to be a 689 pathogen. Nat Rev Microbiol 16:316-328. 10.1038/nrmicro.2018.17. 690 Zipfel, C., and Rathjen, J.P. (2008). Plant immunity: AvrPto targets the frontline. Curr Biol 691 18:R218-220. 10.1016/j.cub.2008.01.016. 692 693 FIGURE LEGENDS 694 Figure 1. Pseudomonas syringae pv. tomato DC3000 polymutants show type III effector-695 dependent phenotypes in Solanum americanum. 696 (A) HR phenotypes triggered by Pto DC3000 wild -type and polymutants. Strains were 697 infiltrated using a needleless syringe (OD 600nm=0.1) into S. americanum accession SP2273 698 leaves. Photographs were taken one day post infiltration. Red dashed borders indicate HR, and 699 black dashed borders indicate no HR within the infiltrated area. (B) In planta bacterial growth 700 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 25 of Pto DC3000 wild-type and polymutants in S. americanum. Bacteria were infiltrated using a 701 needleless syringe at OD 600nm=0.0001 in SP2273. In planta bacterial growth was quantified 702 four days post infiltration. Individual data points are represented by red dots, and the bar s 703 indicate standard deviation. Raw data is presented in Table S4. Statistical significance was 704 determined using one-way ANOV A followed by Dunnett’s multiple tests (ns: nonsignificant, 705 **: p<0.01, ***: p<0.001). GRAPHPAD PRISM v.10.0.1 was used for statistical analysis. (C) 706 Type III effector repertoire s in Pto DC3000 polymutants. Effectors within the box are the 707 remaining effectors in each Pto DC3000 polymutant. 708 709 Figure 2. AvrPto, HopAM1, or HopAD1 triggers a hypersensitive response in Solanum 710 americanum. (A) 11 effectors present in D18E but not in D29E were transiently expressed in 711 SP2273. Agrobacterium carrying C-terminal HA-tagged effectors was infiltrated into SP2273 712 at OD600nm=0.4 with P19 (OD600nm=0.2), which is suppressor of gene silencing . Photographs 713 were taken four days post infiltration. Red borders indicate the presence of HR. (B) Protein 714 accumulation of effectors was confirmed by immunoblot assay. Agrobacterium carrying C-715 terminal HA-tagged type III effectors and P19 were co-infiltrated into Nicotiana benthamiana 716 leaves. The inoculum concentration of the effector was OD 600nm=0.4 and P19 concentration 717 was OD 600nm = 0.2. Leaf samples were collected two days post infiltration. Protein 718 accumulation was detected using an anti-HA antibody. Yellow asterisks indicate expected 719 protein bands. Ponceau S staining shows an equal amount of protein loading. 720 721 Figure 3. Deletion of avirulence effectors enhance s in planta bacterial growth but 722 maintains hypersensitive response induction. (A) Names of Pto DC3000 mutants and 723 deleted effectors. Effector deletions were performed sequentially , as detailed in the Methods 724 section. (B) Pto DC3000 mutants deleted with HR-triggering effectors show enhanced growth 725 compared to wild-type. Bacterial strains were infiltrated at OD600nm=0.0001 using a needleless 726 syringe into SP2273. In planta bacterial growth was assessed 4 days post infiltration. Each red 727 dot represents the individual replicate , with the bar s indicating the standard deviation. Raw 728 data are shown in Table S6. Statistical significance was determined using one -way ANOV A 729 followed by Dunnett ’s multiple tests (ns: nonsignificant, **: p<0.01, ****: p<0.0001). 730 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 26 GRAPHPAD PRISM v.10.0.1 was used for statistical analysis. (C) Pto DC3000 mutants still 731 trigger HR. Bacterial strains were infiltrated at OD 600nm=0.1 using a needleless syringe into 732 SP2273 leaves. HR was scored one day post infiltration on a scale from 0 (no HR) to 7 (full 733 HR), represented as violin plots. HR scoring criteria are shown in Figure S2, revised from the 734 previous study (Ahn et al ., 2023). Individual data points are shown as red dots. Statistical 735 significance was conducted using the Kruskal -Wallis test followed by Dunn ’s multiple 736 comparison test (ns: nonsignificant, **: p<0.01, ****: p<0.0001), using GRAPHPAD PRISM 737 v.10.01. Raw data of HR scoring are shown in Table S7. (D) Representations of HR phenotypes. 738 Bacterial strains were syringe-infiltrated at OD 600nm=0.1 into SP2273 leaves. The HR photos 739 were taken one day post infiltration. The red border indicates the presence of HR. 740 741 Figure 4. HopAA1-1, AvrE1, HopC1, or HopM1 triggers hypersensitive response in 742 Solanum americanum. (A) 18 effectors were transiently expressed in S. americanum leaves. 743 Agrobacterium carrying HA-tagged type III effector and P19 constructs which is suppressor of 744 gene silencing were co-infiltrated into SP2273 at OD 600nm=0.4 and 0.2, respectively . 745 Photographs were taken four days post infiltration. (B) Protein accumulation of effectors was 746 confirmed by immunoblot assay. Agrobacterium carrying C -terminal HA -tagged type III 747 effectors and P19 were co -infiltrated in Nicotiana benthamiana leaves. The inoculum 748 concentration of the effector was OD600nm=0.4, and P19 concentration was OD600nm = 0.2. Leaf 749 samples were collected two days post infiltration. Protein accumulation was visualized using 750 an HA antibody. Yellow asterisks indicate the expected protein size bands. Ponceau S staining 751 shows an equal amount of protein loading. 752 753 Figure 5. Pto DC3000 mutants lacking HR-triggering effectors show enhanced bacterial 754 growth and reduced hypersensitive response phenotypes. (A) Table lists mutant names and 755 deleted effectors in each mutant. Effector deletions were performed sequentially and detailed 756

for effector deletion is explained in Methods section. (B) PKSG 7826, lacking avrPto, 757 hopAM1, hopAD1, hopC1, and hopAA1-1 shows enhanced growth compared to Pto DC3000 758 wild-type. Bacterial strains were infiltrated at OD600nm=0.0001 using a needleless syringe into 759 SP2273 leaves. In planta bacterial growth was quantified four days post infiltration. Individual 760 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 27 data points are represented by red dots, with bars indicating the standard deviation. Statistical 761 analysis was conducte d using one -way ANOV A followed by Dunnett ’s multiple comparison 762 tests (ns: nonsignificant, **: p<0.01, ****: p<0.0001). GRAPHPAD PRISM v.10.0.1 was used 763 for statistical tests. Raw data of bacterial growth are shown in Table S8. (C) PKSG 7892 , 764 lacking all HR-triggering effectors, does not trigger HR. Bacterial strains were infiltrated at 765 OD600nm=0.1 using a needleless syringe into SP2273 leaves. HR was scored one day post 766 infiltration on a scale from 0 (no HR) to 7 (full HR), represented as violin plots. HR scoring 767 criteria are shown in Figure S2, revised from the previous study (Ahn et al., 2023). Individual 768 data points are shown as red dots. Statistical significance was conducted using the Kruskal -769 Wallis test followed by Dunn’s multiple comparison test (ns: nonsignificant, **: p<0.01, ****: 770 p<0.0001), using GRAPHPAD PRISM v.10.01. The raw data of HR scoring are shown in Table 771 S9. (D) Representations of HR phenotypes. Bacterial strains were infiltrated at OD 600nm=0.1 772 using a needleless syringe into SP2273 leaves. The HR photographs were taken one day post 773 infiltration. The red solid borders indicate the presence of HR, and the red dashed borders 774 indicate weak HR. 775 776 Figure 6. PKSG 7826 causes visible disease symptoms in Solanum americanum. (A) 777 Bacterial speck phenotype was caused by PKSG 7826. The bacterial inoculum was infiltrated 778 using a needleless syringe into SP2273 leaves . The concentration of bacterial inoculum was 779 OD600nm=0.00001. Photographs were taken 7 days post infiltration. Pto DC3000 wild-type was 780 used as a negative control. The red border indicates the bacterial disease phenotype and the red 781 dashed border indicates a weak disease phenotype in the infiltrated area. (B) PKSG 782 6 782 induces the bacterial speck in SP2273. The leaves were dipped and swirled in the bacterial 783 inoculum for 2 minutes. The OD600nm of bacterial inoculum was 0.001 and mixed with 0.05 % 784 silwet L-77. The photographs were taken 12 days after dipping. 785 786 Figure 7. Conservation of hypersensitive response-triggering effectors in Pseudomonas 787 syringae strains. (A) Each effector sequence was compared to the reference effector sequences 788 from Pto DC3000. ‘Non-expressed’ effectors are categorized based on expression information 789 from a previous study (Laflamme et al., 2020). ‘Absent’ indicates effectors with an e -value 790 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint 28 higher than 1e -24 or coverage below 60 %. ‘Truncated’ means each strain’s effector coverage 791 compared to the Pto DC3000 is between 60-90 %. ‘Present’ means each strain’s effector and 792 Pto DC3000 effector sequence showed over 90 % identity. The number below the table 793 indicates the percentage of strains number having ‘present’ effector genes out of a total of 117 794 strains. (B) Number of P . syringae strains number according to their effector presence. The 795 color information is the same as the index in Figure 7A. ‘P’ indicates Pseudomonas and ‘Ps’ 796 indicates Pseudomonas syringae. 797 798 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint Figure 1. Pseudomonas syringae pv. tomato DC3000 polymutants show type III effector-dependent phenotypes in Solanum americanum. D18E DC3000 D36E D29E (A) Pto DC3000 wild-type HopAA1-1 HopAA1-2 HopAO1 AvrE1 HopC1 HopD1 HopR1 HopG1 HopM1 HopN1 HopH1 HopQ1-1 HopF2 HopT1-1 HopU1 HopV1 HopX1 HopO1-1 HopE1 HopI1 HopB1 HopAF1 AvrPto HopK1HopAM1 HopY1 AvrPtoB HopA1 HopAD1 D18E HopS2 HopT2HopO1-3’ HopT1-2’HopO1-2 HopS1’ HopBM1 D29E (B) (C) D36E D29E D18E WT 0 1 2 3 4 5 6 7 in planta bacterial growth Log10(CFU/cm2) ns ns *** (A) HR phenotypes triggered by Pto DC3000 wild-type and polymutants. Strains were infiltrated using a needleless syringe (OD600nm=0.1) into S. americanum accession SP2273 leaves. Photographs were taken one day post infiltration. Red dashed borders indicate HR, and black dashed borders indicate no HR within the infiltrated area. (B) In planta bacterial growth of Pto DC3000 wild-type and polymutants in S. americanum. Bacteria were infiltrated using a needleless syringe at OD600nm=0.0001 in SP2273. In planta bacterial growth was quantified four days post infiltration. Individual data points are represented by red dots, and the bars indicate standard deviation. Raw data is presented in Table S4. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple tests (ns: nonsignificant, **: p<0.01, ***: p<0.001). GRAPHPAD PRISM v.10.0.1 was used for statistical analysis. (C) Type III effector repertoires in Pto DC3000 polymutants. Effectors within the box are the remaining effectors in each Pto DC3000 polymutant. .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint EV HopAD1 HopAM1 HopY1 HopAF1 AvrPtoB AvrPto HopK1 HopB1 HopE1 HopI1 HopA1 (A) (B) Ponceau S α-HA 100 70 55 40 kDa * * * * * * * * ** * Figure 2. AvrPto, HopAM1, or HopAD1 triggers a hypersensitive response in Solanum americanum. (A) 11 effectors present in D18E but not in D29E were transiently expressed in SP2273. Agrobacterium carrying C-terminal HA-tagged effectors was infiltrated into SP2273 at OD600nm=0.4 with P19 (OD600nm=0.2), which is suppressor of gene silencing. Photographs were taken four days post infiltration. Red borders indicate the presence of HR. (B) Protein accumulation of effectors was confirmed by immunoblot assay. Agrobacterium carrying C-terminal HA-tagged type III effectors and P19 were co-infiltrated into Nicotiana benthamiana leaves. The inoculum concentration of the effector was OD600nm=0.4 and P19 concentration was OD600nm = 0.2. Leaf samples were collected two days post infiltration. Protein accumulation was detected using an anti-HA antibody. Yellow asterisks indicate expected protein bands. Ponceau S staining shows an equal amount of protein loading. .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint D36E WT PKSG 4673 PKSG 7065 PKSG 7903 PKSG 7377 avrPto X X X X X hopAM1-1 X X X X hopAM1-2 X X X hopAD1 X X Remaining effectors X (A) WT PKSG 4673 PKSG 7065 PKSG 7903 PKSG 7377 D36E (D) (B) (C) D36E WT PKSG 4673 PKSG 7065 PKSG 7903 PKSG 7377 **** 0 1 2 3 4 5 6 7 in planta bacterial growth Log10(CFU/cm2) **** ns ns ns ns 0 1 2 3 4 5 6 7 8 9 10Cell death score D36E WT PKSG 4673 PKSG 7065 PKSG 7903 PKSG 7377 Figure 3. Deletion of avirulence effectors enhances in planta bacterial growth but maintains hypersensitive response induction. (A) Names of Pto DC3000 mutants and deleted effectors. Effector deletions were performed sequentially, as detailed in the Methods section. (B) Pto DC3000 mutants deleted with HR-triggering effectors show enhanced growth compared to wild-type. Bacterial strains were infiltrated at OD600nm=0.0001 using a needleless syringe into SP2273. In planta bacterial growth was assessed 4 days post infiltration. Each red dot represents the individual replicate, with the bars indicating the standard deviation. Raw data are shown in Table S6. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple tests (ns: nonsignificant, **: p<0.01, ****: p<0.0001). GRAPHPAD PRISM v.10.0.1 was used for statistical analysis. (C) Pto DC3000 mutants still trigger HR. Bacterial strains were infiltrated at OD600nm=0.1 using a needleless syringe into SP2273 leaves. HR was scored one day post infiltration on a scale from 0 (no HR) to 7 (full HR), represented as violin plots. HR scoring criteria are shown in Figure S2, revised from the previous study (Ahn et al., 2023). Individual data points are shown as red dots. Statistical significance was conducted using the Kruskal-Wallis test followed by Dunn’s multiple comparison test (ns: nonsignificant, **: p<0.01, ****: p<0.0001), using GRAPHPAD PRISM v.10.01. Raw data of HR scoring are shown in Table S7. (D) Representations of HR phenotypes. Bacterial strains were syringe-infiltrated at OD600nm=0.1 into SP2273 leaves. The HR photos were taken one day post infiltration. The red border indicates the presence of HR. **** **** **** **** .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint HopV1 HopX1 HopT1-1 HopU1HopF2 HopG1 HopO1-1 HopR1 HopN1 HopM1 HopQ1-1HopH1 HopAO1 HopAA1-2 HopAA1-1 AvrE1 EV HopD1 HopC1 (A) 18 effectors were transiently expressed in S. americanum leaves. Agrobacterium carrying HA-tagged type III effector and P19 constructs which is suppressor of gene silencing were co-infiltrated into SP2273 at OD600nm=0.4 and 0.2, respectively. Photographs were taken four days post infiltration. (B) Protein accumulation of effectors was confirmed by immunoblot assay. Agrobacterium carrying C-terminal HA-tagged type III effectors and P19 were co- infiltrated in Nicotiana benthamiana leaves. The inoculum concentration of the effector was OD600nm=0.4, and P19 concentration was OD600nm = 0.2. Leaf samples were collected two days post infiltration. Protein accumulation was visualized using an HA antibody. Yellow asterisks indicate the expected protein size bands. Ponceau S staining shows an equal amount of protein loading. (A) Ponceau S 70 100 40 25 kDa 70 100 40 55 kDa α-HA Ponceau S α-HA * * * * * * * * * * * * * * * * Figure 4. HopAA1-1, AvrE1, HopC1 or HopM1 triggers hypersensitive response in Solanum americanum. .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint (A) D36E WT PKSG 7377 PKSG 7768 PKSG 7826 PKSG 7899 PKSG 7900 PKSG 7892 avrPto X X X X X X X hopAM1-1 X X X X X X X hopAM1-2 X X X X X X X hopAD1 X X X X X X X hopC1 X X X X X X hopAA1-1 X X X X X shcM-hopM1 X X X shcE-avrE1 X X X Remaining effectors X 0 1 2 3 4 5 6 7 8 9 10Cell death score D36E WT PKSG 7377 PKSG 7768 PKSG 7826 PKSG 7899 PKSG 7900 PKSG 7892 **** ns ** ** ** **** **** WT PKSG 7377 PKSG 7768 PKSG 7826 PKSG 7899 D36E PKSG 7900 PKSG 7892 Figure 5. Pto DC3000 mutants lacking HR-triggering effectors show enhanced bacterial growth and reduced hypersensitive response phenotypes. (A) Table lists mutant names and deleted effectors in each mutant. Effector deletions were performed sequentially and detailed method for effector deletion is explained in Methods section. (B) PKSG 7826, lacking avrPto, hopAM1, hopAD1, hopC1, and hopAA1-1 shows enhanced growth compared to Pto DC3000 wild-type. Bacterial strains were infiltrated at OD600nm=0.0001 using a needleless syringe into SP2273 leaves. In planta bacterial growth was quantified four days post infiltration. Individual data points are represented by red dots, with bars indicating the standard deviation. Statistical analysis was conducted using one-way ANOVA followed by Dunnett’s multiple comparison tests (ns: nonsignificant, **: p<0.01, ****: p<0.0001). GRAPHPAD PRISM v.10.0.1 was used for statistical tests. Raw data of bacterial growth are shown in Table S8. (C) PKSG 7892, lacking all HR-triggering effectors, does not trigger HR. Bacterial strains were infiltrated at OD600nm=0.1 using a needleless syringe into SP2273 leaves. HR was scored one day post infiltration on a scale from 0 (no HR) to 7 (full HR), represented as violin plots. HR scoring criteria are shown in Figure S2, revised from the previous study (Ahn et al., 2023). Individual data points are shown as red dots. Statistical significance was conducted using the Kruskal-Wallis test followed by Dunn’s multiple comparison test (ns: nonsignificant, **: p<0.01, ****: p<0.0001), using GRAPHPAD PRISM v.10.01. The raw data of HR scoring are shown in Table S9. (D) Representations of HR phenotypes. Bacterial strains were infiltrated at OD600nm=0.1 using a needleless syringe into SP2273 leaves. The HR photographs were taken one day post infiltration. The red solid borders indicate the presence of HR, and the red dashed borders indicate weak HR . (B) PKSG 7377 PKSG 7768 PKSG 7826 PKSG 7899 PKSG 7900 PKSG 7892 nsns 0 1 2 3 4 5 6 7 In planta bacterial growth Log10(CFU/cm2) D36E WT **** **** **** **** **** (C) (D) .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint WT PKSG 7826 (B) WT PKSG 4673 PKSG 7065 PKSG 7903 PKSG 7377 PKSG 7768 PKSG 7826 PKSG 7899 PKSG 7900 PKSG 7892 (A) Figure 6. PKSG 7826 causes visible disease symptoms in Solanum americanum. (A) Bacterial speck phenotype was caused by PKSG 7826. The bacterial inoculum was infiltrated using a needleless syringe into SP2273 leaves. The concentration of bacterial inoculum was OD600nm=0.00001. Photographs were taken 7 days post infiltration. Pto DC3000 wild-type was used as a negative control. The red border indicates the bacterial disease phenotype and the red dashed border indicates a weak disease phenotype in the infiltrated area. (B) PKSG 7826 induces the bacterial speck in SP2273. The leaves were dipped and swirled in the bacterial inoculum for 2 minutes. The OD600nm of bacterial inoculum was 0.001 and mixed with 0.05 % silwet L-77. The photographs were taken 12 days after dipping. .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint Total HopAD1 8 0 109 0 0 117 HopAM1 25 2 90 0 0 117 AvrPto 34 0 81 0 2 117 HopC1 14 2 101 0 0 117 HopAA1 70 23 24 0 0 117 HopM1 74 16 26 1 0 117 AvrE1 109 3 5 0 0 117 Present (>90 % coverage) Truncated (60-90 % coverage) Absent (90 % coverage) (B) HR-triggering effectors Figure 7. Conservation of hypersensitive response-triggering effectors in Pseudomonas syringae strains (A) (A) Each effector sequence was compared to the reference effector sequences from Pto DC3000. ‘Non-expressed’ effectors are categorized based on expression information from a previous study (Laflamme et al., 2020). ‘Absent’ indicates effectors with an e-value higher than 1e-24 or coverage below 60 %. ‘Truncated’ means each strain’s effector coverage compared to the Pto DC3000 is between 60-90 %. ‘Present’ means each strain’s effector and Pto DC3000 effector sequence showed over 90 % identity. The number below the table indicates the percentage of strains number having ‘present’ effector genes out of a total of 117 strains. (B) Number of P. syringae strains number according to their effector presence. The color information is the same as the index in Figure 7A. ‘P’ indicates Pseudomonas and ‘Ps’ indicates Pseudomonas syringae. .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted May 3, 2025. ; https://doi.org/10.1101/2025.05.01.651788doi: bioRxiv preprint