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Title: A Pathogen Type III Effector with a Novel E3 Ubiquitin Ligase Architecture}
Article:
Abstract:
Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat( LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity.
Introduction:
Most Gram-negative pathogenic bacteria implement the type III secretion system( T3SS) that injects a set of proteins, termed effectors( T3E), directly into the eukaryotic host cell. The effectors' combined function is to subvert the host immune system and to promote bacterial colonization[1],[2]. Plant immunity relies on recognition of conserved pathogen-associated molecular patterns( PAMPs)[3], such as flagellin or bacterial elongation factor Tu[4],[5]. This defense barrier is termed PAMP-triggered immunity( PTI), is activated upon PAMP recognition at the cell surface by specific receptors, followed by a network of cellular signaling events, such as mitogen-activated protein kinase( MAPK) cascades, that ultimately lead to changes in gene expression[3],[6],[7]. In contrast, type III effectors manipulate plant cell processes, often leading to subversion of plant immune responses[1],[8]. T3Es interfere with key eukaryotic cell functions, such as the cytoskeleton rearrangement[9], transcriptional regulation[10],[11] or ubiquitination[12],[13]. However, the biochemical function of the majority of T3Es remains elusive. Ubiquitination is a highly conserved eukaryote-specific post-translational protein modification involving attachment of ubiquitin to the epsilon amine of a lysine residue in the target protein. This modification alters protein activity, protein localization or targets the protein for 26S-proteasome-mediated degradation[14]. Ubiquitination of target proteins involves coupling of ubiquitin to an ubiquitin activating enzyme( E1), transfer to a conjugating enzyme( E2), before an ubiquitin ligase( E3) mediates ubiquitin transfer from an E2 to a target protein[15]. E3 enzymes exhibit high target specificity and differ in the subset of E2s they interact with. Eukaryotic E3s fall into two major classes according to the mechanism of ubiquitin transfer: RING/U-box and HECT domain proteins[16]. RING finger/U-box proteins transfer ubiquitin directly from the E2 to the target protein, whereas HECT proteins first form a thioester intermediate with ubiquitin before ligating it to the target. While ubiquitination is absent in prokaryotes, it emerges as a prime eukaryotic host target for bacterial pathogens, which have evolved diverse T3Es to mimic ubiquitination-related functions. In particular, several bacterial T3Es from animal and plant pathogens function as E3 ubiquitin ligases, represented on one hand by the Pseudomonas syringae T3E AvrPtoB[12],[13] and the NleG family of E. coli T3Es[17], which contain typical U-box folds, and on the other hand by the NEL( novel E3 ligase) domains found in the IpaH and SspH2 T3Es of Shigella and Salmonella spp., respectively[18],[19]. The latter contain a novel thioester-forming E3 ligase domain with no structural homology to the HECT domain. This suggests that during co-evolution with their hosts, pathogenic bacteria have employed different solutions to fulfill the otherwise typical eukaryote-specific function of E3 ubiquitin ligases. Here, we characterized the T3E XopL( Xanthomonas outer protein L) from the model plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria( Xcv), which causes disease on tomato and pepper plants. Xcv injects a suite of ∼30 T3Es into the host cell including the TAL( transcription activator-like) effector AvrBs3, which manipulates plant transcription[10], and the SUMO( small ubiquitin modifier) protease XopD[20]. XopL is a newly identified T3E from Xcv, and was found to exhibit E3 ubiquitin ligase activity. Crystal structure determination revealed that the protein contains a novel fold and thus represents a new class of E3 ubiquitin ligases.
Discussion:
In this study, we identified XopL as a new T3E in Xcv that induces cell death in N. benthamiana and inhibits PTI-related defense gene expression. According to our data, XopL exhibits a robust E3 ubiquitin ligase activity. This activity is associated with its C-terminal region and is required for induction of plant cell death. All ubiquitin ligases known to date including bacterial T3Es with E3 ligase activity belong to the RING/U-box or catalytic( HECT-like) class[16]. RING/U-box proteins act by transferring ubiquitin from E2 directly onto the target protein. T3Es of this class include AvrPtoB from the plant pathogen P. syringae[13], and E. coli NleG[17]. Both T3Es lack significant sequence similarity with RING/U-box proteins but adapt a protein fold similar to that of U-box proteins. On the other hand, the catalytic HECT E3 ligases first attach ubiquitin from the E2 to a catalytic cysteine residue via a thioester intermediate before ligating it to the target protein. A similar mechanism has been adopted by effector proteins of the IpaH and SopA families of animal pathogens[40],[41]. The IpaH and SopA crystal structures are distinct from HECT proteins except for the presence of a catalytic cysteine and certain features of the active site. As XopL lacks cysteine residues in its C-terminal domain, termed XL-box, we hypothesize that it acts by directly transferring ubiquitin from E2 onto a target protein. This is reminiscent of RING/U-box proteins; however, XopL lacks any structural similarity to these E3 ligases. We found that XopL interacts in vitro through its XL-box with a specific family of E2 enzymes, represented by human UBE2D2 and Arabidopsis AtUBC11 and AtUBC28. In Arabidopsis thaliana, AtUBC11 and AtUBC28 are members of the group VI family of E2 enzymes[34]. Many of the 8 family members are ubiquitously expressed in Arabidopsis( including AtUBC28 and AtUBC11) and the three most highly expressed members of this family( AtUBC8, AtUBC10 and AtUBC28; www. genevestigator. com) share 97% sequence similarity with each other. Homologues to these proteins are also found in tomato( S. lycopersicum gi|350536447|; 97% identical to AtUBC28) and pepper( C. annuum gi|40287554|; 96% identical to AtUBC28). Mutation analyses of AtUBC28 revealed amino acid residues F62 and A96 to be critical for the interaction with the XopL E3 ligase. It is worth noting that residue F62 is essential for E2 interactions with HECT E3 ligases[42], but not for interactions with specific RING/U-box proteins[43]. On the other hand, residue A96 in E2 enzymes was shown to contribute to interactions with both HECT- and RING-type ligases plus the bacterial effector SspH2[44]. While this data reveals some molecular details of the XopL interaction with E2 enzymes it cannot be modeled according to previously characterized E3-E2 pairs and requires further structural analysis. XopL-mediated polyubiquitin chains with preponderance of K11 linkages were detected using both Arabidopsis group VI E2 enzymes and the human UBE2D2 enzyme. Ubiquitin contains seven lysine residues that can participate in target protein ubiquitination. Which specific lysine is used is dictated by different E3-E2 enzyme combinations and may trigger different outcomes for a given target protein. Linkage at K48 usually directs target proteins to the proteasome[45], whereas K63-ubiquitination can play a role in signal transduction[46]. The importance of other ubiquitin linkages for cell processes came to light only recently and their physiological role remain largely unknown[47]. A recent report suggested that mixed K11- and K63-linked chains are a virus-internalization signal[48]. In addition, K11-linked ubiquitin chains have been connected to degradation of substrates of the anaphase-promoting complex in cell cycle regulation[49],[50]. The Salmonella T3E E3 ubiquitin ligase SspH2, which similarly to XopL selectively interacts with the human UBE2D2 enzyme, mediates the formation of primarily K48-linked polyubiquitin chains[44]. Considering the predominance of K11-linked polyubiquitination in the case of the interaction between XopL and UBE2D2 or its plant homolog we speculate that K11-linked ubiquitin chains may play an important role in plant-pathogen interactions. However, this remains to be elucidated. Our structural data confirmed that XopL harbors a bona fide LRR domain. The LRR domain is a common feature between XopL and the IpaH- and SspH2- effector E3 ubiquitin ligases mentioned above. While the LRR domain in IpaH plays a regulatory role by inhibiting the E3 activity in the absence of the substrate[18],[19],[51], there is no indication for this kind of mechanism in the case of XopL, as E3 ligase activity is robust in the presence or absence of the LRR domain. However, we were surprised to find that the LRR is involved in suppression of PAMP-elicited gene expression, which we performed using the well-established Arabidopsis protoplast system. According to our data the expression of pNHL10 following elicitation of protoplasts with either flg22 or elf18 peptides was suppressed by the LRR domain, similarly to full-length XopL. This argues for an adaptor function of the LRR domain in which the LRR domain binds a target downstream of PAMP-receptor binding and either downstream or independent of MAPK cascade-signaling, leading to altered gene expression. These results are reminiscent to those reported for the Pseudomonas type III effector AvrPtoB, where suppression of plant immunity by blocking downstream signaling through BAK1-kinase are due exclusively to the two binding domains localized to residues 121–205 and 270–359[52]. As shown by our in planta ubiquitination profiles, the presence of both the LRR and XL-box domains is essential for XopL-induced reactions. While expression of the XL-box domain in planta resulted in formation of additional polyubiquitin chains, in line with its in vitro activity, only full-length XopL with an intact LRR domain triggered cell death. In addition, expression of the individual XL-box and LRR domain had the opposite effect on expression of the NHL10 promoter, even in the absence of PAMP-response elicitor. This suggests that the LRR domain functions as a protein-protein interaction module necessary for both the suppression of PAMP-elicited gene expression and the cell death phenotype we observed. Thus, we hypothesize that XopL fulfills multiple functions in planta by( i) suppressing PTI via its LRR-region and( ii) ubiquitinating a yet unknown plant substrate( s) whose initial recognition may also require the LRR-region. In conclusion, characterization of the bacterial pathogen effector XopL uncovered a novel E3 ubiquitin ligase fold that is part of the pathogen repertoire to mimic an otherwise strictly eukaryotic function such as ubiquitination. This underlines the variety of E3 ligases evolved in pathogenic bacteria for subverting host biology. The next challenge is the identification of host targets of XopL that are involved in suppression of plant defenses, as well as determination of the mechanism of action of this unusual E3 ligase.
Numerous bacterial pathogens infecting plants , animals and humans use a common strategy of host colonization , which involves injection of specific proteins termed effectors into the host cell . Identification of effector proteins and elucidation of their individual functions is essential for our understanding of the pathogenesis process . Here , we identify a novel effector , XopL , from Xanthomonas campestris pv . vesicatoria , which causes disease in tomato and pepper plants . We show that XopL suppresses PAMP-related defense gene expression and further characterize XopL as an E3 ubiquitin ligase . This eukaryote-specific function involves attachment of ubiquitin molecule ( s ) to a particular protein targeted for degradation or localisation to specific cell compartments . Ubiquitination processes play a central role in cell-cycle regulation , DNA repair , cell growth and immune responses . In the case of XopL this activity triggers plant cell death . Through structural and functional analysis we demonstrate that XopL contains two distinct domains , one of which demonstrates a novel fold never previously observed in E3 ubiquitin ligases . This novel domain specifically interacts with plant ubiquitination system components . Our findings provide the first insights into the function of a previously unknown XopL effector and identify a new member of the growing family of bacterial pathogenic factors hijacking the host ubiquitination system .