The Medicago truncatula E3 Ubiquitin Ligase PUB1 Interacts with the LYK3 Symbiotic Receptor and Negatively Regulates Infection and Nodulation[W][OA]

Partner specificity in legume-rhizobia symbiosis involves perception of rhizobial signals by plant lysin motif receptor-like kinases (LysM-RLKs) leading to the formation of nitrogen-fixing root nodules. This work describes PUB1, a protein interactor of LYK3 LysM-RLK, which is involved in regulating the specificity of interaction between Medicago truncatula and Sinorhizobium meliloti

Cell death induction activity of MtNFP-sYFP2 truncated/mutated variants in <i>Nicotiana</i> leaves.

*<p>-see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065055#pone.0065055-Lefebvre1" target="_blank">[22]</a>, PM-plasma membrane, ER-endoplasmic reticulum.</p><p>The designated constructs were expressed alone or co-expressed with <i>MtLYK3-mCherry</i> construct in <i>Nicotiana</i> leaves. Macroscopic symptoms of cell death were scored 48 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented as a fraction of total infiltrations performed. # - In the 3 remaining leaf regions, co-expression of <i>MtNFP</i>[S67A]-<i>sYFP2</i> and <i>MtLYK3-mCherry</i> constructs resulted in increased staining with Evans blue in the entire infiltrated region.</p

Cell death upon MtNFP and MtLYK3 co-production in <i>Nicotiana</i> leaves does not require <i>Sm</i>NF.

<p><i>Agrobacterium</i> transformants carrying either <i>MtNFP-3xFLAG</i> or <i>MtLYK3-3xFLAG</i> construct were co-infiltrated into <i>Nicotiana</i> leaves at a final concentration: OD₆₀₀ [<i>MtNFP</i>] = 0.25 and OD₆₀₀ [<i>MtLYK3</i>] = 0.4 (1); OD₆₀₀ [<i>MtNFP</i>] = 0.15 and OD₆₀₀ [<i>MtLYK3</i>] = 0.25 (2). Twelve hai parts of the transformed regions were syringe-infiltrated with 10⁻⁷mM <i>Sm</i>NF (circled in red) or DMSO diluted to the same concentration (circled in white). Macroscopic observation (left panel) and Evans blue staining (right panel) are depicted 33 hai. Bar is 1 cm.</p

Cell death induction upon (co-)expression of various RLK-encoding genes in <i>Nicotiana</i> leaves.

<p># − unless stated differently: with <i>-3xFLAG</i> (*) untagged (**), or <i>−YFP</i>_N (***) tagged construct.</p><p>Indicated constructs were expressed alone or co-expressed with either <i>MtNFP</i> or <i>MtLYK3</i> in <i>Nicotiana</i> leaves, and the infiltrated regions were marked. Macroscopic symptoms of cell death were scored 48 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented as a fraction of total infiltrations performed.</p

Co-production of MtNFP and MtLYK3 induces cell death in <i>Nicotiana</i> leaves.

<p>A, The following <i>MtNFP</i> and <i>MtLYK3</i> constructs were expressed alone or co-expressed in <i>Nicotiana</i> leaves: mock infiltration (1); <i>MtNFP</i> untagged+<i>MtLYK3</i> untagged (2); <i>MtNFP-sYFP2</i>+<i>MtLYK3-sYFP2</i> (3); <i>MtLYK3-sYFP2</i> (4); <i>MtLYK3</i> untagged (5); <i>MtNFP-sYFP2</i> (6); <i>MtNFP</i> untagged (7). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 48 hai. Bar is 1 cm. B, <i>MtDMI2-sYFP2</i> construct was expressed alone or co-expressed with either <i>MtNFP-mCherry</i> or <i>MtLYK3-mCherry</i> construct in <i>Nicotiana</i> leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 48 hai. Bars are 1 cm.</p

MtNFP and MtLYK3, or AtCERK1 (co-)production in <i>Nicotiana</i> leaves induces defence-like responses.

<p>A, Kinetics of cell death development in <i>Nicotiana</i>. <i>Agrobacterium</i> transformants carrying either <i>MtNFP-3xFLAG</i> or <i>MtLYK3-3xFLAG</i> construct were co-infiltrated into <i>Nicotiana</i> leaves at five different time points (1–5). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 42 hai (region 1), 39 hai (region 2), 36 hai (region 3), 33 hai (region 4) and 30 hai (region 5). Mock infiltration (region 6) was done concomitantly with the infiltration of region 1. Bar is 1 cm. B, Changes in leaf autofluorescence upon MtNFP and MtLYK3 co-production. Leaf regions co-producing MtNFP-3xFLAG and MtLYK3-3xFLAG fusions were analyzed between 24 and 48 hai (here depicted 36 hai) using a stereoscope. Note the decrease in chlorophyll content, as indicated by the decrease of far-red autofluorescence of chlorophyll (left panel), and enhanced accumulation of blue light-excited autofluorescence (right panel) within the infiltrated region. Bar is 1 cm. C, Accumulation of phenolic compounds. The following fusions were (co-)produced in <i>Nicotiana</i> leaves: MtNFP-3xFLAG (1); MtLYK3-3xFLAG (2); MtNFP-3xFLAG+MtLYK3-3xFLAG (3); MtNFP-3xFLAG+MtLYK3[G334E]-3xFLAG (4); AtCERK1-3xFLAG (5); or AtCERK1[K349]-3xFLAG (6). Macroscopic observations (left panel) and subsequent UV-excited autofluorescence of ethanol/lactophenol-cleared (right panel) leaf regions are depicted 36 hai (except for 5–30 hai). Bars are 1 cm. D, Induction of <i>NbHIN1, NbPR1 basic</i>, <i>NbACRE31</i>, and <i>NbACRE132</i> expression in response to separate production or co-production of: MtNFP-3xFLAG (NFP), MtLYK3-3xFLAG (LYK3), MtLYK3[G334E]<i>-</i>3xFLAG (LYK3[G334E]), and AtCERK1-3xFLAG (CERK1). Leaf samples were collected 24 hai and induction of gene expression was analyzed using qRT-PCR. Histograms represent induction of <i>NbHIN1</i> (white columns), <i>NbPR1 basic</i> (grey columns), <i>NbACRE31</i> (hatched columns), and <i>NbACRE132</i> (black columns) normalized by one reference gene, <i>MtEF1 α</i>. Induction of each gene was normalized to that caused by mock infiltration, and then calculated as % induction relative to the induction observed upon co-production of MtNFP and MtLYK3 fusions. Bars represent standard deviation of the mean. At least two technical replicates from two biological replicates were analyzed.</p

Lanthanum chloride delays the cell death development upon MtNFP and MtLYK3, or AtCERK1 (co-)production.

<p><i>Agrobacterium</i> transformants carrying the following constructs were (co-)infiltrated at a final concentration: OD₆₀₀ [<i>MtNFP-3xFLAG</i>] = 0.125 and OD₆₀₀ [<i>MtLYK3-3xFLAG</i>] = 0.2 (A, B); OD₆₀₀ [<i>AtCERK1-3xFLAG</i>] = 0.2 (C, D). Twelve hai parts of the infiltrated regions were syringe-infiltrated with 5 mM lanthanum chloride (circled in red) or water (circled in white). Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 42 hai for leaf regions co-producing MtNFP and MtLYK3 fusions (A, B), and 33 hai for leaf regions producing AtCERK1 fusion (C, D). Cell death development was scored 42 hai (A, B) or 33 hai (C, D): only infiltrations that showed the lack of tissue collapse and no compromised membrane permeability in the lanthanum chloride- or water-treated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.</p

Cell death induction activity of MtLYK3-sYFP2 mutated variants in <i>Nicotiana</i> leaves.

*<p>-see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065055#pone.0065055-KlausHeisen1" target="_blank">[20]</a>, except for the T480A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065055#pone.0065055.s002" target="_blank">Fig. S2</a>), **-see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065055#pone.0065055-KlausHeisen1" target="_blank">[20]</a>, except for the P87S <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065055#pone.0065055-Smit1" target="_blank">[12]</a>, K464A and T480A (this study; number of plants nodulated/number of plants tested).</p><p>The designated constructs were expressed alone or co-expressed with <i>MtNFP-mCherry</i> construct in <i>Nicotiana</i> leaves. Macroscopic symptoms of cell death were scored 48 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented as a fraction of total infiltrations performed. # - despite the lack of pronounced macroscopic symptoms, the co-expression of <i>MtLYK3</i>[K464A]-<i>sYFP2</i> and <i>MtNFP-mCherry</i> constructs resulted in increased staining with Evans blue in the entire infiltrated region.</p

Production of AtCERK1 in <i>Nicotiana</i> leaves induces cell death that requires AtCERK1 kinase activity.

<p><i>AtCERK1-sYFP2</i> (A) and <i>AtCERK1</i>[K349E]-<i>sYFP2</i> (B) constructs were expressed in <i>Nicotiana</i> leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 36 hai. Macroscopic symptoms of cell death were scored 36 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.</p

Structure-Function Similarities between a Plant Receptor-like Kinase and the Human Interleukin-1 Receptor-associated Kinase-4*

Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general