Date Published: August 11, 2016
Publisher: Public Library of Science
Author(s): Suomeng Dong, Yuanchao Wang, Cyril Zipfel.
Plant pathogens secrete a variety of unique and highly specialized effectors to manipulate host immunity. Only a small proportion of the conserved effector genes are present in the genomes of pathogens across kingdoms, providing us with good opportunities to study plant immunity and disease mechanisms. Nucleoside diphosphate-linked moiety X (Nudix) effectors, a group of secreted proteins containing the Nudix hydrolase domain, have been identified in a broad range of pathogens including bacteria, oomycetes, and fungi. Nudix effectors have been validated as pathogenesis players in a few host–pathogen systems, indicating that an important virulence strategy might be shared by distinct plant pathogens. Although progress has been made, many questions remain yet unsolved. Here, we summarize our current knowledge of Nudix effectors. We will also provide thoughts and discussions for the next phase of research.
The nucleoside diphosphate-linked moiety X (Nudix) hydrolases constitute a superfamily of proteins that are ubiquitous in both prokaryotic and eukaryotic organisms . The superfamily members are normally characterized by the presence of a conserved Nudix box (GX5EX7REUXEEXGU, U is usually Ile, Leu, or Val, X is any residue), which catalyzes degradation of nucleoside diphosphate-X (NDP-X) to nucleoside monophosphate (NMP) and phosphate-X (P-X) . Studies in model organisms showed that Nudix proteins perform a variety of functions to sense and modulate levels of their substrates like nucleotide sugars, deoxyribonucleoside triphosphate (dNTPs), and capped mRNAs to maintain proper cellular processes and physiological homeostasis [2,3]. An appealing observation to plant pathologists is that Nudix proteins act as regulators in plant immunity. Arabidopsis AtNUDT7, a well-studied Nudix protein, was initially identified as a pathogen-responsive gene [4,5]. Atnudt7 knockout mutants showed constitutive expression of defense-related genes and accumulation of higher levels of salicylic acid, resulting in enhanced basal defense against bacteria pathogen Pseudomonas syringae and oomycete pathogen Hyaloperonospora parasitica . Investigations demonstrated that AtNUDT7 negatively regulates EDS1 signaling pathway, one of the key components in plant immunity . Interestingly, AtNUDX8, another Nudix protein, was a positive regulator of plant defense. Knockout mutants displayed repressed salicylic acid signaling and significantly enhanced susceptibility to pathogens . Therefore, plant Nudix proteins appear as important players in the plant–pathogen battlefield.
Nudix effectors have been reported in plant pathogenic oomycetes, fungi, and bacteria so far, suggesting that this category of effectors might be important virulence components in the “toolbox” of plant pathogens. We illustrate a schematic view of six representative Nudix effectors varied in structure and size (Fig 1). The expression of Hpx26 effector from R. solanacearum is orchestrated with many other T3SS effectors and the secretion of Hpx26 was verified [8,10]. In Xanthomonas campestris pv. vesicatoria, T3SS Nudix effector XCV0537 was identified . Our BLAST search revealed that the Hpx26 and XCV0537 homologs are widely presented in different Ralstonia and Xanthomonas species. HopAG1, a T3SS effector with putative Nudix domain in Pseudomonas syringae pv. syringae isolate B728a, was previously reported , whereas HopAG1 orthologs were pseudogene in many P. syringae isolates, including DC3000 [13,14]. Moreover, a Nudix effector, namely CtNUDIX, was experimentally validated in fungal Colletotrichum truncatum . Genome sequencing of Colletotrichum pathogens resulted in the identification of one putative Nudix effector from the C. graminicola genome, three from C. higginsianum, two from C. orbiculare, and two from C. fructicola. The genome of Magnaporthe oryzae, the rice blast fungus, also revealed two duplicate copies of putative Nudix effectors [15,16]. However, BLAST searches did not yield CtNUDIX homologous proteins in the genomes of selected phytopathogenic fungi, including Puccinia graminis, Ustilago maydis, Sclerotinia sclerotiorum, and Verticillium dahliae . In oomycetes, PsAvr3b employs a conserved Arg-X-Leu-Arg (RXLR) host-targeting motif, which is responsible for effector entry into host cells [17,18]. Furthermore, both Phytophthora ramorum and Phytophthora capsici genomes encode three putative RXLR-type Nudix effectors. In the Irish potato famine pathogen Phytophthora infestans, however, Nudix effector gene seems to have undergone gene expansion—a total of five RXLR-type and two non-RXLR-type Nudix effectors have been identified [9,15]. Two RXLR-like Nudix effectors, HaRxLL79b/c, were highly induced during infection of H. parasitica . However, we did not harvest Nudix effectors in the necrotrophic oomycete Pythium ultimum and the fish pathogen Saprolegnia parasitica.
Although a broad range of plant pathogens employs Nudix effectors, the biological roles of these Nudix effectors remain poorly understood. Independent studies reported that T3SS Nudix effectors from X. campestris and R. solanacearum are induced during infection of host plants like many other T3SS effectors [8,11]; however, the biological functions of bacterial Nudix effectors have not been reported.
In humans and yeast, many Nudix proteins like Dcp2 are associated with protein complexes to perform functions [20–22]. Recently, it has been reported that the Nudix effector PsAvr3b recruits a host prolyl-peptidyl isomerase (PPIase) cyclophilin protein to modify itself properly for full function . It is shown that PsAvr3b interacts directly with soybean cyclophilin GmCYP1 in vivo and in vitro, and GmCYP1 in turn activates the hydrolase activity of PsAvr3b in a PPIase activity-dependent manner, possibly by isomerizing PsAvr3b into a proper conformation. The substitution of PsAvr3b putative Glycine-Proline (GP) motif, which is a known binding site in cyclophilin substrates, impaired the interaction of PsAvr3b with GmCYP1. Consequently, this mutant can no longer be activated by GmCYP1 and is unable to promote Phytophthora infection or trigger Rps3b-mediated soybean defense response . In summary, this report demonstrated that the cyclophilin protein activates the enzymatic activity of PsAvr3b as a “helper” in host cells to promote PsAvr3b function. Whether other Nudix effectors require additional host factors for function is of interest for further investigation.
The most striking question that remains unclear is the biological functions of Nudix effectors. Beside conserved Nudix domain, the structures and sequences vary greatly among Nudix effectors from different kingdoms, indicating biological functions of these effectors might be different. Although in vitro preferred substrates of PsAvr3b were determined, the natural substrates and the in vivo Nudix enzymatic functions for Nudix effectors as well as their relationship with plant immunity remain poorly understood. Secondly, the protein localization pattern usually provides good information for effector activity. Nudix proteins localize in distinct subcellular components including P-body, peroxisome, and cytosol, which are important to their functions [22,24]; however, little is known for Nudix effectors. Scanning Nudix effectors did not reveal any robust subcellular localization signature; cell biology approaches will be required to investigate Nudix effector subcellular localization. To investigate Nudix effector functions further, a third alternative is to identify binding proteins. Whether these effectors recruit host helper protein to function properly, or what host immune proteins do effectors target to, deserves further exploration.