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1 Manipulation of the host endomembrane system by bacterial effectors

3 Hyelim Jeon1,2 and Cécile Segonzac1,2,3,4*

5 1Department of Agriculture, Forestry and Bioresources, Seoul National University,

6 Seoul, 08826, Republic of Korea

7 2Plant Immunity Research Center, Seoul National University, Seoul 08826, Republic

8 of Korea

9 3Plant Genomics and Breeding Institute, Seoul National University, Seoul, 08826,

10 Republic of Korea

11 4Agricultural and Life Science Research Institute, Seoul National University, Seoul,

12 08826, Republic of Korea

13 *Corresponding author: csegonzac@snu.ac.kr

14

15 Abstract

16 The endomembrane system, extending from the nuclear envelope to the plasma

17 membrane, is critical to the plant response to pathogen infection. Synthesis and

18 transport of immunity-related proteins and antimicrobial compounds to and from the

19 plasma membrane are supported by conventional and unconventional processes of

20 secretion and internalization of vesicles, guided by the cytoskeleton networks.

21 Although plant bacterial pathogens reside mostly in the apoplast, major structural and

22 functional modifications of the endomembrane system in the host cell occur during

23 bacterial infection. Here, we review the dynamics of these cellular compartments,

24 briefly for their essential contributions to the plant defense responses and in parallel,

25 for their emerging roles in bacterial pathogenicity. We further focus on Pseudomonas

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26 syringae, Xanthomonas spp. and Ralstonia solanacearum type III secreted effectors

27 that localize to and/or associate with components of the host endomembrane system

28 or the cytoskeleton network to highlight the diversity of virulence strategies deployed

29 by bacterial pathogens beyond the inhibition of the secretory pathway.

30

31 Keywords

32 Type III secreted effectors, endoplasmic reticulum, vesicle trafficking, secretion,

33 immunity

34

35 Introduction

36 As all eukaryotic cells, plant cells display complex organization, relying on the finely

37 tuned compartmentalization of essential processes for development, growth and

38 survival of the whole organism. Despite technical limitations, mostly imposed by the

39 rigid wall that surrounds and connects the plant cells, plant scientists have adapted

40 and developed state-of-the-art methodologies to provide a modern view of the specific

41 internal structures and processes of the plant cell (Kang et al. 2022). At the heart of

42 the compartmentalization, the endomembrane system is a dynamic membrane

43 structure encompassing the outer nuclear membrane, the endoplasmic reticulum (ER),

44 the Golgi apparatus, the tonoplast, and the multitude of vesicles transporting proteins,

45 lipids and other molecules between these compartments and the periphery of the cell

46 constituted by the plasma membrane (PM) and the cell wall (Figure 1). The

47 endomembrane system contributes to the synthesis of proteins and lipids and is

48 essential for the processing, sorting and delivery of cellular components to and from

49 the vacuole and the PM/apoplast. These processes are tightly and specifically

50 controlled in response to pathogen invasion (Kwon et al. 2008; Wang and Dong 2011).

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51 Invasive pathogens (viral and filamentous) cause major morphological and

52 structural changes in the infected cells. Virus replication and spreading modify protein

53 synthesis and processing in the ER and the ER/PM contact sites at the

54 plasmodesmata (Medina-Puche and Lozano-Duran 2022; Reichel et al. 1999; Wang

55 et al. 2017). The formation of haustoria, the feeding structures of fungi and oomycetes,

56 induces profound remodeling of membranes, including redirection of the

57 endosomal/secretory pathways to form the perihaustorial membrane, changes its lipid

58 composition, and internalizes secreted effectors for the delivery of nutrient (Bozkurt et

59 al. 2015; Bozkurt and Kamoun 2020; Oliveira-Garcia et al. 2021; Rausche et al. 2020).

60 In accordance, plethora of viral proteins and filamentous pathogen effectors target ER-

61 mediated processes or vesicle trafficking (Fabro 2022; He et al. 2020; Jing and Wang

62 2020; Patarroyo et al. 2013; Verchot and Pajerowska-Mukhtar 2021). Although

63 bacterial pathogens mostly multiply in the apoplast, major cytoplasmic changes

64 support the bacterial proliferation (Xin et al. 2016; Xin et al. 2018).

65 Most phytopathogenic bacteria that cause major crop diseases rely on type III

66 secreted effectors (T3Es) for pathogenicity. The T3Es are virulence factors, deployed

67 for manipulating host cell processes to establish a favorable environment (Bundalovic-

68 Torma et al. 2022; Macho 2016; Timilsina et al. 2020). This includes the suppression

69 of the host defense systems, adjustments of water and nutrient contents in the

70 apoplast or modifications of host cell growth and differentiation (Lovelace and Ma 2022;

71 Xin et al. 2018). These host processes are directly or indirectly regulated by the

72 secretory/trafficking pathways of the endomembrane system to ensure changes of the

73 cell wall composition, secretion of antimicrobial compounds and defense proteins,

74 targeting of immune receptors and transporters to the PM and even the regulation of

75 transcription. The impairment of vesicle trafficking and of surface-localized immune

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76 receptor function leads to dysbiosis in the phyllosphere (Chen et al. 2020), providing

77 a clear illustration of the importance of endomembrane dynamics for maintaining

78 balanced interactions with bacterial communities on the plant surface.

79 In this review, we summarize our current understanding of the endomembrane

80 system dynamics during bacterial infection in plants. We also discuss the diverse

81 functions of the T3Es that associate with endomembranes and the underlying

82 cytoskeleton to highlight the contribution of these virulence factors to the development

83 of bacterial disease symptoms.

84

85 Modifications of the endomembrane system during bacterial infection

86 The ER is a large membrane-enclosed compartment shaped by connected tubular

87 (tubules) and flatten sac (cisternae) structures. The overall morphology of the ER

88 varies in the development and the environment of the cell. These changes in the ER

89 structure reflect changes in the ER functionality. ER shaper proteins such as reticulons

90 and LUNAPARK modify and maintain the tubules and the cisternae, respectively.

91 Other ER shaper proteins are required to maintain ER-organelle contact sites,

92 formation of ER-derived compartments such as the autophagosome, or specialized

93 ER domains such as the ER bodies (Kriechbaumer and Brandizzi 2020). Major

94 alterations of the cytoskeleton network and the ER are observed after bacterial

95 infection and are in part imputable to the host defenses. Increased synthesis and

96 secretion of defense proteins and antimicrobial compounds depend on the remodeling

97 of the ER and enhanced and/or re-routed vesicle trafficking. In parallel, recent studies

98 also suggest that some structural changes to the endomembrane system are the

99 results of virulence factor activities and are necessary to promote bacterial pathogen

100 establishment in the apoplast.

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101

102 Modifications controlled by plants as a defense strategy

103 The presence of bacteria in plant apoplast is monitored by a network of surface-

104 localized pattern-recognition receptors (PRRs) belonging to the receptor-like kinase or

105 receptor-like protein families (Boutrot and Zipfel 2017). The PRRs extracellular domain

106 binds to specific microbe-associated molecular patterns (MAMPs), such as fragments

107 of the bacterial flagellin or peptidoglycan and transduces this signal to the cell through

108 the transmembrane domain. As for most integral membrane proteins, PRR synthesis

109 takes place in the ER, where proper folding and glycosylation occur. The newly

110 synthesized PRRs are inserted in the PM following the conventional secretion pathway

111 through the Golgi apparatus, the trans-Golgi network/early endosome and secretion

112 vesicles (Ben Khaled et al. 2015). Upon ligand binding, PRRs are internalized through

113 clathrin-mediated endocytosis (Mbengue et al. 2016). This process contributes to

114 immune signaling and leads to the degradation of activated receptors (Ben Khaled et

115 al. 2015). In turn, immune signaling initiated at the cell surface induces the synthesis

116 of diverse antimicrobial peptides and defense proteins, secreted in the apoplast to

117 ward-off invaders (Campos et al. 2018; Yu et al. 2020). Insertion of callose synthases

118 in the PM further leads to the reinforcement of the cell wall through callose deposition

119 (Wang et al. 2021). Similar to PRRs, these diverse defense components traffic mostly

120 through the conventional secretion pathway, highlighting the essential contribution of

121 the endomembrane system to plant immune responses (Gu et al. 2017; Rosquete and

122 Drakakaki 2018; Wang and Dong 2011).

123 Beside the global enhancement of the secretory pathway activity, multiple

124 studies have also uncovered diverse forms of unconventional secretion pathways that

125 participate in plant defense responses to bacteria (Figure 1). For example, a marked

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126 accumulation of multivesicular bodies (MVBs) is observed in Arabidopsis cells upon

127 Pseudomonas syringae pv. tomato (Pst) infection (Wang et al. 2014). MVBs (also

128 referred to as late endosomes) are usually responsible for delivery of endocytosed

129 receptors to the vacuole for degradation (Hu et al. 2020). The biogenesis of MVBs is

130 regulated by components of the endosomal sorting complex required for transport

131 (ESCRT), which dissociate from membranes through the activation of the AAA

132 ATPase SKD1 stimulated by LYST-INTERACTING PROTEIN 5 (LIP5) (Haas et al.

133 2007). The hypersusceptibility to Pst infection and impaired accumulation of MVBs at

134 the cell periphery in lip5 mutant support the role of MVBs in unconventional secretion

135 of defense compounds (An et al. 2006; Ruano and Scheuring, 2020; Wang et al. 2014).

136 The fusion of MVBs with the PM serves as a major source of extracellular vesicles

137 (EVs) of which accumulation is also observed in the apoplast of cells infected by

138 bacteria (Rutter and Innes 2018; Rybak and Robatzek 2019). EVs deliver proteins,

139 nucleic acids and lipids to the apoplasts and their contribution to cross-kingdom RNA

140 interference has recently been demonstrated (Cai et al. 2021). EVs could also derive

141 from the fusion of the double-membraned exocyst-positive organelles (EXPO) with the

142 PM (Cui et al. 2020; Wang et al. 2010). In accordance, several subunits of the exocyst

143 complex, which mediate the tethering and fusion of vesicles with the PM, are required

144 for immune signaling and their accumulation has been recently observed in response

145 to invasive pathogen infection (Brillada et al. 2021; Kulich et al. 2018; Overdijk et al.

146 2020; Pecenkova et al. 2011; Stegmann et al. 2012).

147 Last but not least, other pathways linked with the endomembrane system

148 including the unfolded protein response (UPR) and autophagy are involved in plant

149 immunity. The contributions of these processes to the plant defense response and to

150 the programed cell death induced in response to recognized strains of pathogens are

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151 still under investigation but are comprehensively reviewed elsewhere (Leary et al.

152 2019; Pastor-Cantizano et al. 2020; Sertsuvalkul et al. 2022; Simoni et al. 2022).

153 Considering the multiple roles in monitoring and defense, the endomembrane

154 compartment emerges as a target of choice for bacterial pathogenicity.

155

156 Modifications induced by bacterial pathogens as a virulence strategy

157 Tracking both the ER morphology (with the luminal RFP-HDEL marker) and the

158 establishment of a Pst strain expressing the fluorescent reporter GFP in the apoplast

159 early after infection, Breeze et al. report a rapid condensation of the ER network in

160 “knot-like structures” preceding the disappearance of the polygonal tubular network in

161 the plant cells in contact with bacterial colonies (Breeze et al. 2020). As these changes

162 are not observed in cells treated with the MAMPs flg22 or chitin and are reminiscent

163 of the ER structural changes observed at penetration sites during filamentous

164 pathogens infection, the authors propose that ER remodeling is a conserved virulence

165 strategy, likely mediated by T3Es and aimed at redirecting the secretion pathway

166 (Breeze et al. 2020; Micali et al. 2011; Takemoto et al. 2003).

167 Another evidence of the manipulation of the ER structure by bacterial

168 pathogens comes from a recent study of the ER bodies. ER bodies can be visualized

169 as relatively large fusiform domains of the ER observed in roots and cotyledons of

170 young plants from the Brassicaceae family. The formation of ER bodies is controlled

171 by the bHLH transcription factor NAI1 and the ER shaper protein NAI2 (Matsushima

172 et al. 2004; Yamada et al. 2008). Although ER bodies are constitutively present in

173 seedlings, their formation can be induced by wounding in mature leaves. Constitutive

174 and induced ER bodies contain sets of beta-glucosidase enzymes (BGLUs)

175 responsible for the breakdown of different glucosinolates. In particular, the activation

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176 of PYK10 (BGLU23) results in the hydrolysis of indole-glucosinolates that are involved

177 in response to herbivory or microbial pathogens (Nakano et al. 2014; Yamada et al.

178 2011). Arabidopsis infection with Pst DC3000 induces the transcription of NAI1 and

179 PYK10, as well as the formation of ER bodies in a type III secretion system- and

180 coronatine-dependent manner (Rufian et al. 2021). Intriguingly, although the presence

181 of ER bodies positively regulates the resistance to herbivory through the facilitated

182 release of toxic glucosinolate derivatives, the formation of ER bodies is prevented

183 during pattern-triggered immunity (Nakazaki et al. 2019; Rufian et al. 2021).

184 Accordingly, treatment with flg22 reduces the number of observable ER bodies in

185 GFP-HDEL expressing plants and the nai1 and pyk10 bglu18 mutant plants are more

186 resistant to Pst DC3000 infection. Therefore, these authors propose that coronatine-

187 induced ER bodies are part of a virulence strategy of Pst to inhibit the plant defense.

188 However, it remains to be determined whether this is a collateral effect of coronatine,

189 a mimic of jasmonic acid, the known inducer of ER bodies, and if T3Es are involved in

190 the induction of ER body formation as suggested by the absence of ER bodies after

191 infection with type III secretion system deficient strain Pst hrcC- (Rufian et al. 2021).

192 Pst infection also induced changes in cytoskeleton networks that can be linked

193 to inhibition of secretion or the establishment of disease symptoms (Henty-Ridilla et

194 al. 2013; Lee et al. 2012; Shimono et al. 2016). Depolymerization of the actin network

195 by Latrunculin B promotes the growth of Pst DC3000 (Henty-Ridilla et al. 2013; Kang

196 et al. 2014). In Arabidopsis leaf pavement cells, Pst DC3000 neither the effector-

197 deficient mutant D28E nor hrcC- leads to increased actin bundling and decreased

198 filament occupancy. This correlates with the onset of chlorosis (Shimono et al. 2016).

199 On the other hand, the disruption of the microtubule network by oryzalin treatment

200 promotes the growth of Pst DC3000 but not that of hrcC-, suggesting that the function

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201 of microtubules in defense compounds secretion is targeted by T3Es as a virulence

202 strategy (Boutté et al. 2007; Crowell et al. 2009; Lee et al. 2012). However, the impact

203 of actin or microtubule network disturbance on the structure of the endomembrane

204 system remains to be examined.

205 Multiple strains of P. syringae, R. solanacearum and X. campestris can produce

206 the phytohormone auxin and/or modulate auxin signaling during infection (Kunkel and

207 Harper 2018). The regulation of plant developmental processes by auxin is in part

208 dependent on the polarized secretion and insertion of the auxin transporters in the PM

209 (Marhava 2022) and indeed, high auxin levels affect endocytosis and endomembrane

210 dynamics (Narasimhan et al. 2021). Hence, it is possible that infection with auxin-

211 producing bacterial pathogens modifies vesicle trafficking, resulting in developmental

212 modifications, such as those observed in R. solanacearum infected Arabidopsis roots,

213 which include early differentiation of root hair cells and xylem vessels and are

214 dependent on the auxin receptor TIR1 (Lu et al. 2018; Naseem et al. 2015; Xue et al.

215 2022).

216 As better methods and techniques are being established, our view of the

217 bacterial infection processes at the cellular level improves. The aforementioned

218 studies bring robust evidence for the manipulation of the endomembrane system as a

219 virulence strategy of bacterial pathogens to suppress plant defenses or alter plant

220 physiology. Although the mechanisms remain elusive, these modifications likely result

221 from the activities of T3Es that target components of the endomembrane system

222 and/or the associated cytoskeleton network.

223

224 Bacterial effectors target the endomembrane system.

225 T3Es localize to or associate with the endomembrane system.

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226 The subcellular localization of T3Es is commonly inferred from imaging of effector-

227 fluorescent protein (FP) fusions heterologously expressed with cellular markers or

228 fluorescent dyes (Aung et al. 2017). This approach could be complemented using a

229 split-FP system to directly visualize T3Es trafficking upon natural delivery (Henry et al.

230 2017; Li et al. 2014; Park et al. 2017). Together these live imaging methods,

231 sometimes backed up by ultrastructural studies, have comforted the view that bacterial

232 T3Es can localize to various cellular compartments following their delivery in the

233 cytoplasm, including the PM and the endomembrane system (Figure 1 and Table 1)

234 (Block et al. 2014; Nomura et al. 2011; Schulze et al. 2012; Xin et al. 2015). However,

235 as fluorescence microscopy remains constrained by resolutive limitations and protein

236 expression/accumulation, indications of T3Es targeting the endomembrane system

237 are also brought forward by biochemical characterization of the T3E target proteins in

238 the host cell.

239 Large-scale protein-protein interaction studies using yeast-two-hybrid (Y2H),

240 immunoprecipitation/mass spectrometry or more recently proximity-labeling with biotin,

241 have revealed the extent of the possible reach of T3Es in plant cells (González-Fuente

242 et al. 2020; Khan et al. 2018a; Mukhtar et al. 2011). Further curation of the T3E

243 interactor lists could be used to infer T3E subcellular localization (Khan et al. 2018b).

244 Interestingly, these studies highlighted the prominence of PM proteins as putative

245 targets for bacterial T3Es, and also revealed a notable association with the ER and

246 the endosomal compartments (Khan et al. 2018b). Although T3Es can have multiple

247 (yet uncharacterized) targets, it is noteworthy that significant Gene Ontology terms

248 associated with T3E-interacting proteins are related to the cytoskeleton, protein

249 processing or protein folding and stability (Khan et al. 2018b). Furthermore, these

250 large-scale studies uncovered the convergence of multiple effectors on hub proteins,

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251 hence categorized as important nodes of the plant immune system (González-Fuente

252 et al. 2020; Mukhtar et al. 2011). Two such hub proteins, the exocyst subunit EXO70E2

253 and the MVB biogenesis regulator LIP5, belong to the endomembrane system,

254 contribute to immunity and interact several different R. solanacearum T3Es in Y2H

255 (González-Fuente et al. 2020; www.effectorK.org; Reddit et al. 2019; Wang et al.

256 2014). Other R. solanacearum T3Es, including the ER-localized RipD, are also found

257 to interact with proteins from the Prenylated Rab Acceptor PRA1 family which are

258 involved in protein trafficking in the ER and protein transport from the Golgi (González-

259 Fuente et al. 2020; Lee M.H. et al. 2011, 2017; Jeon et al. 2020). The P. syringae T3E

260 HopC1, previously shown to localize to PM, interacts with the exocyst complex

261 component SEC5A and kinesin motor protein in Y2H, which suggests an effect on the

262 secretory pathway (Dowen et al. 2009; Mukhtar et al. 2011). Although the biological

263 roles of these interactions remain to be determined, these data support the relevance

264 of T3Es localization to the endomembrane system.

265

266 T3Es disrupt trafficking and secretion.

267 Multiple studies have highlighted T3E targets involved in vesicle trafficking and protein

268 secretion (Table 1) following the pioneer characterization of the Pst T3E HopM1

269 function in the plant cell (Nomura et al. 2006). HopM1 localizes to the TGN/EE,

270 interacts with the ADP ribosylation factor guanine nucleotide exchange factor (ARF-

271 GEF) HopM1 interactor 7 (MIN7), and mediates MIN7 degradation in a proteasome-

272 dependent manner (Nomura et al. 2006, 2011). Importantly, HopM1 is essential for

273 the full virulence of Pst DC3000 and accordingly, MIN7 is required for flg22-induced

274 callose deposition and salicylic acid (SA)-regulated protein secretion. Interestingly, the

275 establishment of an aqueous apoplast (previously referred to as water-soaking lesions)

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276 mediated by HopM1-MIN7 interaction also plays a crucial role in bacterial infection

277 (Xin et al. 2016). This latest study revealed that HopM1 triggers abscisic acid (ABA)

278 biosynthesis and signaling pathway, promoting stomatal closure and water-soaking

279 lesions (Roussin-Léveillée et al. 2022). These results imply that immune suppression

280 through inhibition of vesicle trafficking by HopM1 could be a side effect of HopM1

281 interference with ABA signaling. Indeed, the consequential aqueous apoplast could

282 potentially dilute antimicrobials or affect cell wall-based defense. However, another

283 line of evidence supports the direct suppression of immunity-associated vesicle

284 trafficking by HopM1. Zhao et al. have shown that MIN7 is critical for cutin formation

285 and surface defense responses against Pst DC3000 (Zhao et al. 2020). Considering

286 that MIN7 is one of ARF-GEFs which are the key components of the vesicle trafficking

287 system, it further highlights the function of MIN7 in defense-associated trafficking (Suo

288 et al. 2021). Similarly, the Pst T3E AvrPto, first identified as a suppressor of cell wall

289 defense, was found to interact with RabE GTPases involved in polarized secretion of

290 proteins from the Golgi apparatus to the PM (Bogdanove and Martin, 2000; Hauck et

291 al. 2003; Nielsen 2020; Speth et al. 2009). Although the specific virulence mechanism

292 is unknown, both T3Es target regulators of trafficking pathways.

293 Deposition of callose in the cell wall is commonly used as an indicator of

294 activation of the plant immune pathway and can be suppressed by multiple T3Es. This

295 assay allows the rapid screening of effector repertoires for immuno-suppressive

296 activity, although the direct impairment of the secretory pathway components required

297 for callose deposition could only be deciphered in a handful of cases. The Pst T3E

298 HopE1 and X. campestris pv. vesicatoria T3E XopB and XopJ were shown to

299 specifically block the secretion a GFP-reporter fused to a signal sequence (Bartetzko

300 et al. 2009; Guo et al. 2016; Schulze et al. 2012). HopE1 actually targets an isoform

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301 of the plant Ca2+-sensor calmodulin to modify the microtubule network supporting

302 vesicle transport as a virulence strategy (Guo et al. 2016). XopJ, located in the PM

303 and Golgi, was later shown to inhibit the proteasome function, which appears to be

304 required for normal secretion (Üstün et al. 2013). XopB localizes to the Golgi or Golgi-

305 derived vesicles and also impairs defense-associated reactive oxygen species (ROS)

306 signaling (Priller et al. 2016; Schulze et al. 2012). Beyond model species studies, three

307 other T3Es from X. axonopodis pv. manihotis were also identified as suppressors of

308 callose deposition in cassava (Medina et al. 2018). Additionally, two T3Es from X.

309 campestris pv. vesicatoria XopE2 and XopF2, localized in PM and Golgi respectively,

310 inhibit callose deposition and contribute to disease symptoms (Popov et al. 2016;

311 Thieme et al. 2007). Links between the subcellular localization of these T3Es and their

312 contribution to bacterial virulence should be further elucidated through the

313 identification and the functional study of their targets in the host cell.

314 Direct links between exocytosis and T3E-mediated suppression of plant

315 immunity were recently uncovered for the Pst T3E AvrPtoB and the X. campestris T3E

316 XopP (Michalopoulou et al. 2022; Wang et al. 2019). Interestingly, both T3Es target

317 the EXO70B subunits of the exocyst complex to interfere with the plant immune

318 system. EXO70B proteins are necessary for MAMP-triggered immunity and required

319 for the recruitment of immunity-related proteins such as the PRR FLAGELLIN

320 SENSING 2 (FLS2) or RESISTANCE TO PSEUDOMONAS SYRINGAE PV

321 MACULICOLA 1-INTERACTING PROTEIN 4 (RIN4) to the PM (Sabol et al. 2017;

322 Stegmann et al. 2012; Wang et al. 2020). Furthermore, exo70b1 mutant displays

323 spontaneous cell death, partially dependent on the activation of the truncated immune

324 receptor TN2, indicating that EXO70B1 integrity is monitored by the immune system

325 (Zhao et al. 2015). AvrPtoB was identified along 5 other Pst T3Es as an interactor of

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326 EXO70B1. AvrPtoB is an active E3-ligase in plant cells, where it mediates the

327 ubiquitination and degradation of EXO70B1 in a proteasome-dependent manner, thus

328 promoting virulence of Pst in Arabidopsis (Wang et al. 2019). On the other hand, no

329 known enzymatic activity could be ascribed to XopP, although the presence of XopP

330 homologs contributes to virulence of Xanthomonas strains in rice and radish (Ishikawa

331 et al. 2014; Jiang et al. 2009). X. campestris XopP associates with several

332 components of the subcomplex II of the exocyst complex and might prevent further

333 intermolecular interactions in the exocyst. Surprisingly, XopP expression affects

334 specifically the stability of the EXO70B1 subunit but does not trigger TN2-mediated

335 resistance (Michalopoulou et al. 2022). Nonetheless, transient and stable expression

336 of XopP significantly impairs FLS2 recruitment to the PM as well as the secretion of

337 the antimicrobial protein PR-1, illustrating the importance of the final steps of

338 exocytosis for the plant defense responses and the clear benefit of T3E targeting this

339 process for pathogenicity.

340

341 T3Es interfere with the cytoskeleton.

342 Accumulating evidence of T3E-cytoskeleton interactions in the recent decade provides

343 mechanistic links with changes in cytoskeleton networks observed during bacterial

344 infection (Table 1). Although these changes have not been linked with the

345 modifications observed in the endomembrane system upon infection, the cytoskeleton

346 roles in supporting and framing internal membranes is clearly established in plant cells

347 (Brandizzi 2021). Among the cytoskeleton interacting T3Es, HopE1 is part of the

348 minimal functional T3E repertoire necessary for Pst DC3000 virulence, supporting the

349 importance of the host cytoskeleton as a target of the pathogens (Cunnac et al. 2011;

350 Xin et al. 2018).

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351 HopW1 from P. syringae pv. maculicola was the first T3E shown to directly

352 target actin (Kang et al. 2014). HopW1 depolymerizes the filamentous actin (F-actin)

353 in plant cells and in vitro. Moreover, HopW1 inhibits actin-dependent endocytosis and

354 the ER/vacuolar protein targeting, while promoting bacterial growth when delivered by

355 Pst DC3000. These results imply that actin-dependent trafficking pathways are

356 important for controlling pathogen growth during infection. Interestingly, the HopW1

357 homolog in Acidovorax citrulli, AopW1 also disrupts the actin network and contributes

358 to virulence (Jiménez-Guerrero et al. 2021; Traore et al. 2019). AopW1 localizes to

359 the ER and early endosomes (EEs) where it alters the ER distribution (Jiménez-

360 Guerrero et al. 2021). Whether AopW1 localization in different endomembrane

361 compartments results in additional effect on actin-dependent pathways remains to be

362 examined.

363 HopE1 is one of the T3Es essential for Pst virulence and is associated with the

364 microtubule network (Cunnac et al. 2011; Guo et al. 2016). HopE1 binds the

365 microtubule-bundling protein MICROTUBULE ASSOCIATED PROTEIN 65-1

366 (MAP65-1) to dissociate it from microtubules. In presence of HopE1, the

367 disorganization of the microtubule network correlates with a defect in secretion of the

368 antimicrobial PR-1 and enhanced plant susceptibility (Guo et al., 2016). Pst also

369 deploys the T3E HopZ1a to interfere with the microtubule network (Lee et al., 2012).

370 HopZ1a localizes to the PM where it acetylates tubulin and leads to a dramatic

371 disruption of cortical microtubule network, resulting in suppression of the secretory

372 pathway and cell wall-mediated defense. These studies indicate that the microtubule

373 network plays a role in plant defense by mediating delivery of antimicrobial molecules

374 or possibly, the release of EVs.

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375 Finally, the X. campestris pv. vesicatoria T3E XopL with E3 ligase activity is

376 presumed to ubiquitinate an unknown target protein(s) associated with microtubule

377 (Erickson et al. 2018; Singer et al. 2013). Recently, a component of the autophagy

378 pathway, SH3 DOMAIN-CONTAINING PROTEIN 2 (SH3P2), was identified as the

379 host target of XopL (Leong et al. 2022). XopL interacts with and mediates proteosomal

380 degradation of SH3P2, which suppresses host autophagy turnover and promotes

381 infection. Whether XopL functions in the microtubule traffic and autophagy

382 independently or whether there is a direct link between these two processes remains

383 elusive. It is noteworthy that SH3P2 is also involved in clathrin-mediated endocytosis

384 (Nagel et al. 2017), which implies that XopL may affect endocytic trafficking via

385 modulation of the microtubule cytoskeleton.

386

387 Endomembrane-associated T3Es suppress plant immunity independent of secretion.

388 Beside the suppression of the secretory pathway or the modifications of the

389 cytoskeleton networks, alternative functions for T3Es associated with the

390 endomembrane system have been uncovered (Figure 1 and Table 1). The Pst

391 DC3000 T3E HopD1 was identified from its ability to suppress HopA1-induced

392 effector-triggered immunity (ETI) in N. tabacum (Guo et al. 2009; Jamir et al. 2004).

393 HopD1-mediated ETI suppression was confirmed in Arabidopsis by co-delivering

394 HopD1 and the avirulent T3E AvrRpm1 (Block et al. 2014). This specific suppression

395 of ETI correlates with HopD1 contribution to Pst virulence. The Arabidopsis

396 membrane-tethered NAC transcription factor NTL9 was identified as a HopD1

397 interactor in a large-scale Y2H screen (Mukhtar et al. 2011). HopD1 and NTL9 interact

398 at the ER, where membrane-tethered transcription factors are held and then, NTL9 is

399 cleaved and released as an active form that can enter the nucleus (Kim et al. 2006).

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400 NTL9 is a positive regulator of plant immunity since the growth of Pst DC3000 wild

401 type and avirulent strains was restricted in Arabidopsis expressing the constitutively

402 active NTL9. On the other hand, in ntl9 mutant, the growth of the Pst DC3000 hopD1

403 mutant was enhanced, which further supports that HopD1 targets NTL9 to suppress

404 ETI. In addition, HopD1 suppresses the expression of NTL9-regulated genes

405 specifically induced during ETI but not PTI. How HopD1-NTL9 interaction at the ER

406 can affect NTL9-dependent transcriptional regulation in the nucleus is yet to be

407 elucidated.

408 While most of R. solanacearum T3Es remain uncharacterized, RipN is the only

409 ER-associated R. solanacearum T3E for which a biological function has been

410 characterized (Sun et al. 2019). RipN contains a Nudix hydrolase domain which

411 confers ADP-ribose/NADH pyrophosphorylase activity in vitro. RipN hydrolase activity

412 causes reduction of NADH/NAD+ ratio but does not affect protein ADP-ribosylation

413 nor the ratio of reduced/oxidized glutathione which is a major indicator of cellular redox

414 status in Arabidopsis inoculated with R. solanacearum (Aon et al. 2007; Zhao et al.

415 2009). Furthermore, expression of RipN but not the catalytically inactive mutant RipN-

416 4Q suppresses MAMP-induced callose deposition and transcription of defense-related

417 genes in Arabidopsis, resulting in enhanced growth of Pst DC3000 and R.

418 solanacearum GMI1000. Hence, RipN contributes to pathogen virulence by

419 manipulating NADH/NAD+ homeostasis. Although RipN hydrolase activity is

420 indispensable for its function, deletion of the Nudix domain does not affect subcellular

421 localization of RipN to the ER and the nucleus. The identification of host targets of

422 RipN will help correlate localization and biological function of RipN to elucidate its

423 virulence mechanism.

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424 In addition to intracellular trafficking, cell-to-cell communication through

425 plasmodesmata (PD) plays critical roles in plant-pathogen interactions and plant

426 defense signaling (Brunkard and Zambrysk 2017; Cheval and Faulkner 2018). PD are

427 membrane-lined channels that connect the PM, ER and cytoplasm of neighboring cells,

428 allowing direct molecular exchange between cells (Cheval and Faulkner 2018; Lucas

429 et al. 2009). Recent findings have shown that PD are targeted by multiple pathogens

430 to promote their spreading to neighboring cells (Kankanala et al. 2007; Khang et al.

431 2010; Redkar et al. 2015; Sakulkoo et al. 2018). For example, the Pst DC3000 T3E

432 HopO1-1 localizes to PM and PD (Aung et al. 2020). Transgenic Arabidopsis

433 expressing HopO1-1 shows increased PD-mediated diffusion of YFP. HopO1-1

434 interacts with and destabilizes PD-localized proteins PDLP5 and PDLP7 which are

435 important regulators of callose homeostasis at PD (Cui and Lee 2016; Lee et al. 2011).

436 Both pdlp5 and pdlp7 Arabidopsis mutants are more susceptible to Pst infection than

437 wild type plants. Moreover, Pst DC3000 hopO1-1 mutant has compromised virulence,

438 indicating that HopO1-1 contributes to virulence by destabilizing PDLPs and altering

439 the intercellular trafficking (Aung et al. 2020; Lee J. Y. et al. 2011). More recently,

440 multiple Pst DC3000 T3Es were observed to move intercellularly, which was inhibited

441 by PD closure (Li et al. 2021). Although local callose deposition at the PD collar affects

442 the aperture of PD, the role of desmotubule (the ER tubule at the center of PDs) in

443 intercellular transport remains to be clarified but could constitute a gateway for effector

444 transport to neighboring cells. These recent findings reveal the importance of PD

445 regulation for both plant defense and pathogen infection and further question the role

446 of the endomembranes supporting the PD structures.

447

448 Concluding remarks and open questions

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449 Dynamics of the endomembranes plays a pivotal role in the timely execution of

450 defense responses and the fine-tuning of pathogen perception components at the cell

451 surface. Recent observations have additionally revealed that the endomembrane

452 compartments can be modified by bacterial pathogens to promote virulence.

453 Endomembrane-localized T3Es and their host targets form a polyvalent interface that

454 has recently come on focus. Besides a deeper understanding of the mechanisms

455 deployed for inhibition of the secretory pathway, the characterization of the

456 endomembrane-associated T3Es also reveals the importance of these compartments

457 for the regulation of transcription, ROS homeostasis, and intra- and/or inter-cellular

458 trafficking. Hence, further characterization of T3E activities on the host targets will

459 improve our current view of plant-bacteria interactions and expand our understanding

460 of the endomembrane-localized processes in the plant cell.

461 Several lines of questioning may frame further investigations on the

462 endomembrane-associated T3Es: i) how are the bacterial T3Es recruited to the

463 endomembrane system after the delivery into the cytoplasm? ii) can we decipher the

464 relationships between cytoskeleton networks modifications and endomembrane

465 functions using T3Es as probes? iii) how could the T3Es target the secretion of specific

466 molecules? Preventing PR1/antimicrobials secretion or callose synthase/PRR

467 insertion in the PM is an efficient strategy to promote bacterial proliferation. However,

468 a general inhibition of the secretory pathways could also prevent the PM insertion of

469 nutrient transporters or modifications of the cell wall beneficial for the pathogen. iv)

470 beside the conventional secretory pathway, other endomembrane-localized processes

471 (ER-QC, UPR, unconventional secretion, and autophagy) contribute to plant immunity.

472 There are numerous examples of bacterial effectors targeting these processes in

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473 animal cells but none has been reported in phytopathogenic bacteria so far (Alshareef

474 et al. 2021).

475

476 Author contributions

477 H. J. and C. S. conceived, prepared and wrote this review.

478

479 Acknowledgements

480 We thank Dr Wanhui Kim and the other members of our laboratory for their comments

481 on our manuscript. We apologize to colleagues whose work could not be cited due to

482 space limitation. This work was supported by the National Research Foundation of

483 Korea (NRF) funded by the Ministry of Sciences and ICT (Project No.

484 2018R1A5A1023599) and by the Ministry of Education (Project No.

485 2022R1I1A1A01066399).

486

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930
931
932 Table 1. Bacterial effectors targeting the plant endomembrane system
Pathogen Effector Target Contribution Activity/function Reference
to virulence
Disruption of actin
Acidovorax citrulli Jiménez-Guerrero et al.
AopW1 F-actin Yes cytoskeleton and ER
(M6, 7a1) 2021; Traore et al. 2019
organization
Pseudomonas
syringae pv. Disruption of actin-
HopW1 F-actin Yes Kang et al. 2014
maculicola dependent vesicle trafficking
(ES4326)
Pseudomonas Acetyltransferase; disruption
syringae pv. HopZ1a Tubulin Yes of microtubule networks, Lee et al. 2012
syringae (A2) inhibition of protein secretion
Pseudomonas Bogdanove and Martin
RabE
syringae pv. AvrPto Yes 2000; Hauck et al. 2003;
GTPase
tomato (DC3000) Speth et al. 2009
Pseudomonas
Guo et al. 2009; Wang
syringae pv. AvrPtoB EXO70B1 Yes E3 ubiquitin ligase
et al. 2019
tomato (DC3000)
Pseudomonas
Inhibition of NTL9-regulated Block et al. 2014; Guo et
syringae pv. HopD1 NTL9 Yes
gene expression during ETI al. 2009
tomato (DC3000)

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Pseudomonas
Calmodulin,
syringae pv. HopE1 Yes Inhibition of protein secretion Guo et al. 2009, 2016
MAP65-1
tomato (DC3000)
Pseudomonas
Proteasome-dependent Nomura et al. 2006,
syringae pv. HopM1 MIN7 Yes
MIN7 destabilization 2011
tomato (DC3000)
Pseudomonas ADP-ribosyltransferase;
PDLP5,
syringae pv. HopO1-1 Yes promotion of PD Aung et al. 2020
PDLP7
tomato (DC3000) permeability
Pseudomonas
SEC5A, Dowen et al. 2009;
syringae pv. HopC1 Unknown Putative cysteine protease
kinesin Mukhtar et al. 2011
tomato (DC3000)
Ralstonia
ADP-ribose/NADH
solanacearum RipN Unknown Yes Sun et al. 2019
pyrophosphorylase
(GMI1000)
Ralstonia
González-Fuente et al.
solanacearum RipD PRA1 Unknown Unknown
2020; Jeon et al. 2020
(GMI1000)
Xanthomonas
Michalopoulou et al.
campestris pv. XopP EXO70B1 Yes Inhibition of exocytosis
2022
campestris (8004)
Xanthomonas
Priller et al. 2016;
campestris pv. XopB Unknown Yes Inhibition of protein secretion
Schulze et al. 2012
vesicatoria (85-10)
Xanthomonas
Cystein protease; inhibition Bartetzko et al. 2009;
campestris pv. XopJ RPT6 Yes
of protein secretion Üstün et al. 2013, 2015
vesicatoria (85-10)
Xanthomonas E3 ubiquitin ligase; Erickson et al. 2018;
campestris pv. XopL SH3P2 Yes suppression of autophagy Leong et al. 2022;
vesicatoria (85-10) and stromules Singer et al. 2013
Xanthomonas
Unknown; inhibition of Popov et al. 2016;
campestris pv. XopE2 Unknown Yes
callose deposition Thieme et al. 2007
vesicatoria (85-10)
Xanthomonas
Unknown; inhibition of
campestris pv. XopF2 Unknown Yes Popov et al. 2016
callose deposition
vesicatoria (85-10)
933

934

935 Figure legend

936 Figure 1. Bacterial effectors associate with the plant endomembrane system.

937 Components of the plant immune system such as the pattern-recognition receptors

938 (PRR), the callose synthase or antimicrobial peptides are delivered to the apoplast

939 through the conventional secretion pathway including the nuclear envelope, the

940 endoplasmic reticulum (ER), the Golgi apparatus, the trans-Golgi network/early

941 endosome (TGN/EE) and associated vesicles that fuse with the plasma membrane

942 (PM) through the activity of the exocyst complex. Immune components can also be

943 encapsulated in extracellular vesicles (EVs), derived from the multivesicular body

944 (MVB) or the exocyst-positive organelle (EXPO). Multiple effectors (T3Es) contributing

945 to bacterial pathogen virulence are found in association with the different

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946 compartments of the endomembrane system or the underlying cytoskeleton (pink

947 boxes) and interfere with plant immunity. This figure was created with BioRender.com.

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Figure 1

254x177mm (600 x 600 DPI)