Functional Analysis of the Type 3 Effector Nodulation Outer Protein L (NopL) from Rhizobium sp. NGR234

Abstract

Rhizobia are nitrogen-fixing bacteria that induce nodules on roots of legume host plants. Nodule initiation is triggered by rhizobial Nod factors, i.e. lipo-chitooligosaccharidic nodulation signals produced in response to host flavonoids in the rhizosphere (1). Additional symbiotic determinants produced by specific rhizobial strains often influence establishment of symbiosis in a host-specific manner. For example, rhizobial effector proteins secreted by bacterial secretion systems can significantly affect nodule initiation and nitrogen fixation on certain host plants (2). Various rhizobial strains possess a type 3 secretion system and deliver type 3 effector proteins into eukaryotic host cells (3, 4), a strategy common to many pathogenic Gramnegative bacteria (e.g. Yersinia spp., Shigella spp., Salmonella spp., and Pseudomonas spp.; Ref. 5).Once delivered into host cells, many type 3 effectors of pathogenic bacteria function as virulence factors, which manipulate the host cell by circumventing or suppressing innate immunity. Type 3 effectors may target the ubiquitination-26 S proteasome pathway, alter RNA metabolism, or interfere with protein kinases in host cells (6 -8). Various type 3 effectors target components of mitogen-activated protein kinase (MAPK) pathways. For example, YopJ of Yersinia spp. inactivates human MAPK kinase by acetylation of amino acid residues in the activation loop (9) and the IpaH 9.8 effector of Shigella spp. is an E3 ubiquitin ligase that targets the MAPK kinase Ste7 in yeast and interferes with nuclear factor B signaling in human cells (10,11). Furthermore, a number of type 3 effectors (HopAI1/OspF/ SpvC family) from plant and animal pathogens inactivate MAPKs by cleaving the C-OP bond of phosphothreonine at their TXY motif (Thr-Xaa-Tyr, where Xaa is any amino acid) in the activation loop (12). MAPK signal pathways are major elements of the innate immunity of the plant and play crucial roles in recognition of pathogens as well as in activation of plant defense responses (13,14). Overexpression of genes involved in MAPK signaling can result in induction of a hypersensitive response (HR), 3 i.e. rapid localized cell death. Overexpression of SIPK (salicylic acid-induced protein kinase) and NtMEK2 (the MAPK kinase upstream of SIPK) induced necrosis of tobacco leaves within a few hours (15)(16)(17)

Mutualistic Co-evolution of Type III Effector Genes in Sinorhizobium fredii and Bradyrhizobium japonicum

“…Rhizobial T3E genes show genetic patterns indicative of surprising conservation, pointedly contrasting the patterns consistent with the dynamic arms race model of co-evolution dogmatic for T3Es (Figures 3–5). This finding is particularly striking in light of the observations that T3Es of mutualistic rhizobia are similar in regards to those of pathogens in having to maintain sufficiency in engaging and dampening PTI while avoiding ETI [15], [17], [24], [47]. Moreover, we demonstrated that the high conservation of T3Es in rhizobia relative to phytopathogens is not likely driven by differences in host range or phylogenetic diversity among genomes (Figures 1, S2, 4, and 5).…”

Use of the Cre-loxP recombination system as an estimate for Agrobacterium-mediated co-transformation of tobacco leaves

“…Simultaneous analysis of two or more proteins in the same plant cell has gained increasing importance. Examples include work on a bacterial type-3 secretion effector (Zhang et al 2011), targeted transgene integration into plant cells (Cai et al 2009), and subcellular localization of proteins (Marion et al 2008). As this approach requires transformation of the same host cell by two bacteria, optimal transformation conditions must be chosen and the percentage of co-transformed cells should be as high as possible.…”

“…Transformation of tobacco leaves is also widely used to study the function of microbial effectors delivered into plant cells (e.g. Dai et al 2008;Krasileva et al 2010;Zhang et al 2011).…”

NopD of Bradyrhizobium sp. XS1150 Possesses SUMO Protease Activity

“…However, besides secretion and translocation into host cells, only a few rhizobial T3 effectors have been biochemically characterized in detail. Examples of well-studied rhizobial effectors are the nodulation outer proteins NopE1/NopE2 (Wenzel et al, 2010;Schirrmeister et al, 2011), NopL (Bartsev et al, 2003;Bartsev et al, 2004;Zhang et al, 2011;Ge et al, 2016), NopM (Rodrigues et al, 2007;Kambara et al, 2009;Xin et al, 2012;Xu et al, 2018), NopP (Ausmees et al, 2004;Skorpil et al, 2005;Zhao et al, 2018;Sugawara et al, 2018), NopT (Dai et al, 2008;Dowen et al, 2009;Kambara et al, 2009;Fotiadis et al, 2012), and ErnA (Teulet et al, 2019).…”

“…The T3 effector NopL of Sinorhizobium sp. (=Ensifer fredii) NGR234, for example, becomes multiply phosphorylated by MAP kinases and thereby inhibits MAP kinase signaling (Zhang et al, 2011;Ge et al, 2016). On the other hand, plants can recognize the presence or action of a specific T3 effector (avirulence protein) by a given intracellular disease resistance protein (nucleotidebinding/leucine-rich repeat receptor).…”