The large, diverse, and robust arsenal of Ralstonia solanacearum type III effectors and their in planta functions

1Bacteria from the Ralstonia solanacearum species complex (RSSC) are soilborne plant pathogens responsible for bacterial wilt on more than 250 species, moko and blood diseases of banana, brown rot of potato, and Sumatra disease on clove trees (Peeters et al., 2013a). Due to its aggressiveness, broad host range, widespread geographical distribution, and long‐lasting persistence on the soil, Ralstonia ranks among the most devastating plant pathogenic bacteria (Mansfield et al., 2012). For a successful infection, RSSC bacteria rely on different virulence determinants, including the production of exopolysaccharides and phytohormones, secretion of cell wall‐degrading enzymes, detoxification, and nutrient‐scavenging systems and motility (Genin and Denny, 2012). However, the main virulence determinant of RSSC bacteria is the type III secretion system (T3SS), a “molecular syringe” that allows the translocation of several type III effector proteins (T3Es) directly into the host cell (Coll and Valls, 2013). These T3Es, referred to as Ralstonia injected proteins (Rips), are able to subvert the defences and modify the metabolism of the host to promote virulence.

2Since the first RSSC T3E genes were cloned in the 1990s (Carney and Denny, 1990; Arlat et al., 1994; Guéneron et al., 2000), different approaches have been conducted to systematically identify at the genome scale the full T3E repertoire of several RSSC strains. Two main strategies were undertaken: (a) sequence‐based approaches, searching for sequence homology with previously described effector genes and/or for the presence of certain 25‐nucleotide cis elements in their promoters, the hrp II box or the plant‐inducible promoter (PIP) box motifs (Salanoubat et al., 2002; Cunnac et al., 2004a; Gabriel et al., 2006; Peeters et al., 2013b; Sabbagh et al., 2019), and (b) regulation‐based strategies, exploiting that T3E gene expression is controlled by HrpB, an AraC family member of transcriptional regulators (Genin et al., 1992; Cunnac et al., 2004a). Regulation‐based strategies include gene expression studies (Cunnac et al., 2004b; Occhialini et al., 2005) and genetic screens using random transposon‐insertion mutagenesis (Mukaihara et al., 2004). Verification of the T3SS‐dependency of the secretion or translocation is typically required to confirm the bona fide T3E status of in silico predicted or candidate T3Es (Lonjon et al., 2018). Most translocation analyses exploit the adenylate cyclase (Cya) reporter system (Cunnac et al., 2004b; Mukaihara and Tamura, 2009; Mukaihara et al., 2010). T3SS‐dependent secretion analyses compare the secreted proteins, detected by immunoblotting or mass spectrometry, of wild‐type compared to hrp mutant strains (Tamura et al., 2005; Solé et al., 2012; Lonjon et al., 2016; Sabbagh et al., 2019).

3A recent genomic study on 140 RSSC strains identified the pan‐effectome of the species complex, consisting of 102 T3E and 16 hypothetical T3E genes (Sabbagh et al., 2019). RSSC strains carry on average 64 T3E genes (minimum 45 in R. syzygii subsp. syzygii strain R24 and maximum 76 in R. pseudosolanacearum strain Rs‐10‐244). This contrasts with other plant pathogenic bacteria such as Pseudomonas syringae and Xanthomonas campestris, with an average of 31 (min. 3, max. 53) and 23 (min. 12, max. 28) T3E genes, respectively (Roux et al., 2015; Dillon et al., 2019). The existence of several paralog families, such as the RipG (former GALA), RipS (SKWP), RipA (AWR), RipH (HLK), or RipP (PopP) families, can be considered as a remarkable feature of the RSSC. Not a single known RSSC strain does not carry multiple copies of these paralog T3E families. This contributes to the large size of the RSSC pan‐effectome. The T3E repertoires of different RSSC strains are quite diverse, with only 16 core T3Es (i.e., T3Es present in at least 95% of sequenced strains), which represents 13.6% of the RSSC pan‐effectome (Sabbagh et al., 2019). This core‐effectome is larger than in P. syringae (four core T3Es, 5.7% of its pan‐effectome) or X. campestris (three core T3Es, 8.6% of its pan‐effectome) (Roux et al., 2015; Dillon et al., 2019). Several studies have tried to connect the T3E diversity to the host specificity of RSSC strains (Ailloud et al., 2015; Cho et al., 2019; Sabbagh et al., 2019). Although some host specificity determinants could be identified, the power of such studies has usually been largely limited by the lack of exhaustive strain host range empirical data.

4As model root and vascular plant pathogens, RSSC bacteria are among the pathogens with a larger number of functionally characterized T3Es. Some effectome‐scale experiments have tried to shed light on the function of RSSC T3Es through systematic determination of their ability to induce a hypersensitive response (HR; Wroblewski et al., 2009), inhibit plant defences (Nakano and Mukaihara, 2019a), or identify their plant targets (González‐Fuente et al., 2020). However, most of our current knowledge on effector function comes from smaller‐scale experiments in which often one or a few T3Es are studied. To date, we have counted more than 50 different RSSC T3Es that have been characterized with varying degrees of detail (Figure 1 and Table 1). One of the main factors complicating this task is the observed genetic redundancy among different RSSC T3Es (Angot et al., 2006; Solé et al., 2012; Chen et al., 2014). This redundancy is likely to ensure a more robust virulence strategy for the bacteria (Ghosh and O’Connor, 2017), although it makes the functional dissection of single effectors more complicated, particularly for the paralog families. Nevertheless, some members of these families can still have specific and nonredundant functions (Angot et al., 2006; Turner et al., 2009; Wang et al., 2016).

5Similar to other pathogens, RSSC T3Es collectively contribute to the pathogen fitness in the plant through different and not always well‐characterized mechanisms (Toruño et al., 2016). These include the interference with the plant basal defence responses, alteration of the plant metabolism, and avoidance of the specific recognition of other T3Es. However, some RSSC T3Es can also be recognized by specific plant genotypes and induce strong immune responses.

6The subversion of basal defences is one of the most studied functions of pathogen effectors. Several RSSC T3Es are known to interfere with different host cellular processes involved in these basal defence responses. RipP2 (former PopP2) relies on its acetyltransferase activity to acetylate the WRKY domain of the plant homonymous transcription factors, which prevents their association with DNA and subsequent expression of defence‐related genes (Le Roux et al., 2015). RipAY is selectively activated by eukaryotic thioredoxins to degrade the host glutathione, which plays an important role in plant immunity (Fujiwara et al., 2016, 2020; Mukaihara et al., 2016; Sang et al., 2018). RipAR and RipAW rely on their E3 ubiquitin ligase activity to inhibit plant defence responses (Nakano et al., 2017). Also linked to ubiquitination, the RipG (former GALA) family of T3Es presents a eukaryotic F‐box domain required for the interaction with Arabidopsis components of the Skp, Cullin, F‐box containing (SCF) complex contributing to Ralstonia virulence (Angot et al., 2006; Remigi et al., 2011). RipAL is a chloroplastic effector with a lipase domain required for the induction of jasmonic acid (JA) production and suppression of salicylic acid (SA) signalling (Nakano and Mukaihara, 2018). The inhibition of SA‐mediated defences seems also to be the role of RipR (former PopS) and RipG1 and RipG3, although the molecular mechanisms behind this inhibition still remain unknown (Jacobs et al., 2013; Medina‐Puche et al., 2019). RipAB (former PopB) down‐regulates the calcium signalling pathway and inhibits the plant basal defences (Zheng et al., 2019). Finally, RipN contains a Nudix hydrolase domain required to alter the NADH/NAD+ ratio in planta and to inhibit the plant defence responses (Sun et al., 2019).

7In addition to these functionally characterized RSSC T3Es, other basal defence inhibiting T3Es have been identified in large‐scale screenings. Sixteen additional RSSC T3Es have been reported as suppressors of the flg22‐induced reactive oxygen species (ROS) production, a marker typically associated with pathogen‐associated molecular pattern (PAMP)‐triggered immunity (Sang and Macho, 2017): RipA5 (former AWR5), RipAD, RipAF1, RipD, RipE1, RipI, RipQ, RipAC (former PopC), RipAL, RipAP, and RipAU; and to a lesser extent RipH1 (former HLK1), RipM, RipS1 (former SKWP1), RipAN, and RipB (Nakano and Mukaihara, 2019a; Jeon et al., 2020).

8Plant pathogenic bacterial T3Es can also interfere with different host metabolic processes to promote the bacterial survival, release nutrients, and facilitate the infection (Macho, 2016). RSSC bacteria thrive in the xylem, manipulating the composition of the xylem sap (Lowe‐Power et al., 2018). This manipulation can occur through different mechanisms, including the T3SS, as RSSC bacteria are able to inject T3Es into living cells surrounding the vasculature (Vasse et al., 2000; Henry et al., 2017). Indeed, some RSSC T3Es display different activities that could modulate the plant metabolism. One of the better characterized examples is RipTAL (former Brg11), which presents homology with Xanthomonas spp. transcription activator‐like (TAL) effectors (de Lange et al., 2013). RipTAL induces the expression of plant genes involved in the synthesis of polyamines, evading their native translational regulation mechanisms (Wu et al., 2019). It is hypothesized that this RipTAL‐induced boost of the plant polyamine levels prevents the proliferation of possible Ralstonia competitors (Wu et al., 2019). RipA5 acts as an inhibitor of the conserved target of rapamycin (TOR) pathway in yeast and plant cells (Popa et al., 2016). As a key regulator of the switch between growth and stress responses (Dobrenel et al., 2016), RipA5‐mediated inhibition of the plant TOR pathway leads to reduced nitrate reductase activity (Popa et al., 2016). Lastly, RipTPS possesses trehalose‐6‐phosphate synthase activity in yeast (Poueymiro et al., 2014). As trehalose‐6‐phosphate is a key regulatory molecule in plant metabolism (Baena‐González and Lunn, 2020), RipTPS could potentially interfere with this regulation but so far this activity has not been shown in planta.

9In addition to the beforementioned RSSC T3Es for which functional roles could be assigned, other T3E genes have been also identified as contributors to bacterial virulence on different hosts. These additional T3E genes have been identified through pathogenicity or competitive index assays with single or multiple gene mutants. These tests allow us to pinpoint the involvement in virulence but do not provide further information about the underlying molecular mechanisms. This is the case for RipA2 and RipD, which contribute to virulence in tomato (Cunnac et al., 2004b), or RipAA and RipG7, important in the early and late stages of infection of the model legume species Medicago truncatula, respectively (Turner et al., 2009; Wang et al., 2016). RipAC, RipAF1, RipAK, RipAV, RipAY, RipD, RipP2, RipR, RipS4, RipY, and RipTAL contribute to bacterial fitness in eggplant (Macho et al., 2010). For RipD and RipP2, this contribution to fitness was also demonstrated in tomato and bean, and in the case of RipAA, exclusively in tomato (Macho et al., 2010). The RipA family members contribute collectively to virulence in both eggplant and tomato (Solé et al., 2012), and the RipH family members also contribute to virulence in tomato (Chen et al., 2014). RipAM, RipAN, and RipBH contribute significantly to virulence in potato (Zheng et al., 2019), and RipAC acts similarly in tomato (Yu et al., 2020).

10Through evolution, plants have evolved mechanisms to recognize specific RSSC T3Es and induce a strong defence response often associated with a hypersensitive response (HR) (Balint‐Kurti, 2019). This is precisely what was observed on petunia with RipX (former PopA), the first RSSC T3E to have been characterized (Arlat et al., 1994). This same phenotype was later observed in tobacco (Belbahri et al., 2002; Racapé et al., 2005), and could be explained by a RipX‐mediated inhibition of the gene expression of the ATP synthase F1 subunit α (Sun et al., 2020). RipAA and RipP1 (former AvrA and PopP1, respectively) trigger strong HRs in diverse Nicotiana spp. (Carney and Denny, 1990; Robertson et al., 2004; Poueymiro et al., 2009; Chen et al., 2018). Additionally, RipP1 also triggers anHR on petunia St40 line (Lavie et al., 2002), and RipAA, in pepper CW300 and RNaKy accessions (Wroblewski et al., 2009). RipP2 was the first RSSC T3E for which the corresponding immune receptor was identified in Arabidopsis: Recognition of R. solanacearum 1 (RRS1) (Deslandes et al., 1998, Deslandes et al., 2003). It was later shown that this recognition also involves the Resistance to Pseudomonas syringae 4 (RPS4) immune receptor (Gassmann et al., 1999; Narusaka et al., 2009; Williams et al., 2014). The RPS4/RRS1‐dependent immunity is activated by RipP2 acetylation of RRS1 C‐terminal WRKY domain representing an integrated decoy that mimics RipP2 virulence targets (Tasset et al., 2010; Le Roux et al., 2015; Sarris et al., 2015). RipAT and RipAV induce HR‐like phenotypes when expressed in most lettuce and certain pepper and tomato cultivars (Wroblewski et al., 2009). RipA1, RipA2, RipA3, and RipA5 trigger HRs with varying intensities on different Nicotiana spp. (Solé et al., 2012; Jeon et al., 2020). RipTPS produces an HR specifically on N. tabacum independently of its enzymatic activity (Poueymiro et al., 2014). RipAX2 (former Rip36) elicits immunity on wild and cultivated eggplants in a Zn‐finger domain‐dependent (Nahar et al., 2014) and independent (Morel et al., 2018) manner, respectively. RipAB triggers an HR in N. benthamiana but only when localized in the nucleus (Zheng et al., 2019). RipB induces chlorosis in different Nicotiana spp. in a Recognition of XopQ1 (Roq1)‐dependent manner (Nakano and Mukaihara, 2019b). RipBN triggers resistance in tomato in a Pseudomonas tomato race 1 (Ptr1)‐dependent manner (Mazo‐Molina et al., 2019). RipE1 triggers immune responses mediated by both SA and JA in N. benthamiana and Arabidopsis (Sang et al., 2020). RipE1 also triggers an HR in N. tabacum and N. benthamiana in a Suppressor of G2 allele of skp1 (SGT1)‐dependent manner for the latter (Jeon et al., 2020). Last, RipI triggers immune responses in tomato and cell death in yeast and N. benthamiana, the latter through interaction with the plant basic helix‐loop‐helix 93 (bHLH93) transcription factor (Deng et al., 2016; Zhuo et al., 2020).

11The recognition of RSSC T3Es and subsequent strong immune responses can also be counteracted through the action of other T3Es, sometimes referred as “meta‐effectors” (Kubori et al., 2010). This could allow the bacteria to conserve effectors with potent virulence functions for which a given host has already developed specific recognition capabilities. This is the case for RipAY, which can inhibit the previously mentioned RipE1‐triggered immunity (Sang et al., 2020). RipAY inhibits RipE1‐mediated activation of the SA signalling pathway probably through degradation of the plant cellular glutathione (Mukaihara et al., 2016; Sang et al., 2018, 2020). It has also been proposed that RipAC suppresses RipE1‐triggered immunity, inhibiting in this case SGT1‐mediated MAPK activation (Yu et al., 2020). RipAK is able to prevent Ralstonia‐induced HR in N. tabacum by inhibiting plant catalase activity (Sun et al., 2017). Whether this HR is induced by RipAA, RipB, and/or RipP1, responsible for RSSC incompatibility in N. tabacum (Poueymiro et al., 2009; Nakano and Mukaihara, 2019b), is still unknown.

12In this microreview, we have summarized the current knowledge about RSSC T3Es. Despite being one of the largest and most studied bacterial plant pathogen effectomes, a majority of RSSC T3Es remain poorly characterized to date. This will undoubtedly change in the near future as more and more RSSC T3Es are currently being characterized by several research groups worldwide. Nevertheless, from what is currently known, we can already see that the large RSSC effectome is highly diversified in terms of molecular functions, subcellular localizations, and host‐targeted processes. RSSC T3Es act in the host plasma membrane, cytoplasm, nucleus, chloroplasts, or peroxisomes, and interfere with the plant gene expression regulation at the transcriptional and translational level, metabolism, ubiquitination, phytohormone production and signalling, redox homeostasis, and calcium signalling. This functional repertoire, coupled with genetic and functional redundancy, confers RSSC bacteria with a strong, varied, and robust set of weaponry against their hosts. It is thus tempting to hypothesize that this T3E diversity contributes to the adaptability of Ralstonia as a species complex to a wide range of plant hosts. It should also be noted that this large cornucopia of T3Es could be a key factor in the appearance of RSSC strains adapted to new host plants, like the recently identified strains virulent on cucurbitaceous crops (Wicker et al., 2007), coffee plant (Lopes et al., 2015), fig tree (Jiang et al., 2016), African daisy (Weibel et al., 2016), and roses (Tjou‐Tam‐Sin et al., 2017). Future work will help to elucidate whether the so far uncharacterized T3Es target similar processes to those previously described or if, on the contrary, they interfere with completely different plant processes. This is key to understanding whether the strength of RSSC effectomes comes from its high diversity (i.e., RSSC bacteria target simultaneously many different plant processes) or from its redundancy (i.e., RSSC bacteria target a few key plant processes with redundant T3Es). The characterization of new T3Es will also allow the plant processes that RSSC bacteria specifically target to be determined to establish a successful infection. Interestingly, 9 out the 16 RSSC core T3Es have been shown to contribute to virulence in different hosts: RipA2, RipAB, RipAM, RipAN, RipAY, RipG5, RipG6, RipH2, and RipR. From these nine T3Es, functional information is only available for five of them: RipG5 and RipG6 interact with components of the E3 ubiquitin ligase complex (Angot et al., 2006; Remigi et al., 2011), RipR inhibits SA‐mediated defence responses (Jacobs et al., 2013), RipAY degrades plant glutathione (Fujiwara et al., 2016, 2020; Mukaihara et al., 2016; Sang et al., 2018), and RipAB down‐regulates the calcium signalling pathway (Zheng et al., 2019). These different processes, together with the unknown ones targeted by the other core T3Es, could represent the minimum plant processes that Ralstonia needs to modulate. This “basal arsenal” could be complemented with accessory T3Es that could have additive effects, targeting the same or different processes. However, this characterization might prove quite complex as these plant processes, and their modulation by Ralstonia T3Es, might vary substantially among different organs and host species. The diverse, and sometimes large, host range of RSSC strains and the functional diversity and redundancy of its effectome are therefore some of the causes of RSSC adaptability and aggressiveness, but also some of the major factors complicating its systematic and exhaustive study. A valuable tool that will open a wide variety of possibilities in the decipherment of RSSC T3E functions is the generation of a strain devoid of all its effectors, as has been performed on the P. syringae strain DC3000 (Cunnac et al., 2011). This should be completed soon on the RSSC strain OE1‐1 (K. Onishi, Kochi University, Japan, personal communication). The fact that RSSC bacteria can infect both model and agronomically important crop species confers a practical perspective to this information gathered over the last decades. This should certainly contribute to the design of effective and sustainable control measures against the devastating RSSC.