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Subfunctionalization of NRC3 altered the genetic structure of the Nicotiana NRC network [1]

['Ching-Yi Huang', 'Institute Of Plant', 'Microbial Biology', 'Academia Sinica', 'Taipei', 'Yu-Seng Huang', 'Yu Sugihara', 'The Sainsbury Laboratory', 'University Of East Anglia', 'Norwich Research Park']

Date: 2024-12

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play crucial roles in immunity against pathogens in both animals and plants. In solanaceous plants, activation of several sensor NLRs triggers their helper NLRs, known as NLR-required for cell death (NRC), to form resistosome complexes to initiate immune responses. While the sensor NLRs and downstream NRC helpers display diverse genetic compatibility, molecular evolutionary events leading to the complex network architecture remained elusive. Here, we showed that solanaceous NRC3 variants underwent subfunctionalization after the divergence of Solanum and Nicotiana, altering the genetic architecture of the NRC network in Nicotiana. Natural solanaceous NRC3 variants form three allelic groups displaying distinct compatibilities with the sensor NLR Rpi-blb2. Ancestral sequence reconstruction and analyses of natural and chimeric variants identified six key amino acids involved in sensor-helper compatibility. These residues are positioned on multiple surfaces of the resting NRC3 homodimer, collectively contributing to their compatibility with Rpi-blb2. Upon activation, Rpi-blb2-compatible NRC3 variants form membrane-associated punctate and high molecular weight complexes, and confer resistance to the late blight pathogen Phytophthora infestans. Our findings revealed how mutations in NRC alleles lead to subfunctionalization, altering sensor-helper compatibility and contributing to the increased complexity of the NRC network.

Plants utilize complex immune systems to fend off invading pathogens. The nucleotide-binding domain and leucine-rich repeat (NLR) proteins function as intracellular immune receptors and play major roles in plant immunity. In solanaceous plants, several sensor NLRs form a complex genetic network with helper NLRs called NRCs (NLR-required for cell death) to trigger immune responses. However, the evolution of genetic compatibility between sensor NLRs and NRCs was unclear. Here, we showed that NRC3, one of the NRC subgroups in solanaceous plants, underwent subfunctionalization after the Solanum-Nicotiana divergence, altering the NRC network in Nicotiana. Using natural, chimeric, and reconstructed ancestral NRC variants, we mapped six critical residues on multiple protein surfaces of NRC3 contributing to subfunctionalization. These findings reveal how mutations in NRC alleles lead to subfunctionalization, altering sensor-helper NLR compatibility and increasing the complexity of plant immune systems.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: JK received funding from industry on NLR biology at the time of the study. JK has filed patents on NLR biology. Other authors have declared that no competing interests exist.

Funding: The work was supported by National Science and Technology Council (NSTC-110-2311-B-001-044, NSTC-111-2628-B-001-023, NSTC-112-2628-B-001-007 to CHW), intramural fund of Institute of Plant and Microbial Biology, Academia Sinica (CHW), National Institute of Agricultural Botany Fellowship (LD), the Gatsby Charitable Foundation (LD, YS, AT, JK), the Royal Society (LD), and BASF Plant Science (JK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we addressed a fundamental feature of the NRC network architecture: what are the molecular determinants of sensor/helper specificity? We focused on the evolution of specificity between NRC3 orthologs to the sensor NLR Rpi-blb2. We found that Rpi-blb2 can only signal (referred to as compatible from here on) through a subset of NRC3 variants. Using ancestral sequence reconstruction, we showed that the change of compatibility evolved through subfunctionalization. We mapped the determinants that affect the compatibility of NRC3 orthologs with Rpi-blb2 to three residues in the NB-ARC domain and three residues in the LRR domain. These residues are positioned at three surfaces on the resting NRC3 homodimer, and collectively contribute to their compatibility with Rpi-blb2. We propose that the NRC network evolved through successive cycles of helper NLR duplications followed by mutations leading to subfunctionalization. These subfunctionalization events may divide the NLR network into smaller subnetworks, increasing the complexity of the immune system.

While NLRs often show high diversity across plant species, many well-studied examples are NLRs that have remained largely conserved throughout evolution. For example, ZAR1, which represents an ancient category of plant immune receptors, indirectly recognizes effectors by engaging with its RLCK (Receptor-Like Cytoplasmic Kinase) partners, forming pentameric resistosome complexes associated with the membrane [ 4 , 12 ]. Similar to ZAR1, helper NLRs, including ADR1, NRG1, and NRCs, form membrane-associated punctate and high molecular weight complexes upon activation by their respective sensor NLRs [ 13 – 18 ]. Recent research has uncovered that the activations of NRCs engage the conformational changes of resting homodimer complexes into hexameric resistosome complexes, providing valuable insights into the regulation of immunity conferred by the NRC network [ 19 – 22 ]. However, the exact mechanism by which the induction of the NRC resistosome complex engages with the coordination of both sensor and helper NLRs, as well as how the compatibility between these sensor-helper NLRs is determined in the network, remains unclear. As the sensor and helper NLRs within the NRC network trace back to a common ancestral sensor-helper cluster similar to other NLR pairs, it is intriguing to observe that diverse levels of compatibility have evolved among multiple helper NLRs and sensor NLRs [ 10 , 11 ].

The NRC networks likely originated from a sensor-helper NLR gene cluster that emerged predating the divergence of asterids and Caryophyllales, and then massively expanded in lamiids, in particular in solanaceous plants and several Ipomoea species [ 9 – 11 ]. Among all the NRC family members described thus far, NRC0 is the only conserved NRC across lineages of asterid plants [ 10 ]. NRC0 orthologs from different species are often located in a gene cluster together with the sensor NLRs that are NRC0-dependent [ 10 , 11 ]. Many of the NRC0 subclade members can function with NRC0-dependent sensor NLRs from plants of other lineages, indicating that NRC0 orthologs are largely conserved and have partially retained their compatibility with sensor NLRs across different species [ 10 , 11 ]. Interestingly, the NRC networks are highly expanded in most lamiids, with several family-specific NRC subclades showing features of diversifying selection [ 11 ]. Most of these family-specific NRC members in lamiids do not function with tested sensor NLRs from different plant families, suggesting that the expansion of NRC networks has led to specialized pairings, resulting in low sensor-helper compatibility with NLRs from distantly related species [ 11 ].

In asterids, the NRC (NLR-required for cell death) family represents a group of helper NLRs functioning downstream of multiple sensor NLRs [ 9 – 11 ]. In solanaceous plants, three of the NRCs, namely NRC2, NRC3, and NRC4, display partial genetic redundancy as well as specificity to different sensor NLRs, resulting in an NLR network with intricate genetic structure. For example, the sensor NLR Rpi-blb2 signals through NRC4 but not NRC2 and NRC3, the sensor NLR Prf signals through NRC2 and NRC3, whereas the sensor NLR Rx can signal redundantly through NRC2, NRC3 or NRC4 in the model solanaceous plant species Nicotiana benthamiana [ 9 ].

NLR (nucleotide-binding domain and leucine-rich repeat) proteins are intracellular receptors used by both plants and animals to detect invading pathogens [ 1 , 2 ]. They usually consist of an N-terminal domain that is essential for initiating downstream responses, an NB-ARC domain that binds ADP or ATP/dATP, and a leucine-rich repeat region that is involved in pathogen recognition [ 2 ]. Typical plant singleton NLRs, such as ZAR1, can recognize pathogen molecules and initiate downstream responses through the formation of a pentameric resistosome complex that functions as a calcium channel [ 3 – 5 ]. However, some NLR proteins have evolved into sensor and helper NLRs that function together, forming pairs or complex networks to confer resistance against invading microbes [ 2 , 6 – 8 ].

Results

NRC3 orthologs and paralogs show different compatibility with the sensor NLR Rpi-blb2 To gain insights into the molecular mechanisms and evolution of the NRC network, we cloned several NRC homologs from tomato and N. benthamiana and performed complementation assays with several sensor NLRs in the nrc2/3/4 CRISPR knockout (nrc2/3/4_KO) N. benthamiana line [23]. We reasoned that exploring closely related NRC homologs that show different compatibilities to the sensor NLRs may shed light on the molecular determination and evolution of the sensor-helper compatibility. The cloned NRCs were grouped into NRC0 to NRCX based on the phylogenetic analysis (S1 Fig) [9,24–26]. We individually expressed these NRCs with Rx, Sw5b, Prf (Pto/AvrPto), Gpa2, Rpi-blb2, and R1 with their corresponding effector proteins in N. benthamiana leaves, and performed cell death intensity quantification using autofluorescence-based imaging (S2A Fig). While most of the sensor-helper genetic dependency results were consistent with the previous report, tomato NRC3 (SlNRC3) but not N. benthamiana NRC3 (NbNRC3) rescued Rpi-blb2-mediated cell death in the nrc2/3/4_KO N. benthamiana (Figs 1A, 1B, and S2B–S2F). To explore the differences among the NRC3 variants, we performed phylogenetic analyses of several NRC3 sequences identified from solanaceous plants. We found that most solanaceous NRC3 homologs are clustered into three allelic groups, including Group A (NRC3a) which contains orthologs of NRC3 from Solanum and Capsicum species, and Group B (NRC3b) and Group C (NRC3c) which contains NRC3 from Nicotiana species (Fig 1C). Both N. tabacum and N. sylvestres harbor sequences of NRC3b and NRC3c, whereas N. benthamiana only contains one NRC3c sequence (Fig 1C). We cloned several of these NRC3 variants and tested their ability to rescue Rpi-blb2, Prf (Pto), and Rx-mediated cell death in the nrc2/3/4_KO N. benthamiana. We found that the two NRC3a variants tested were able to rescue Rpi-blb2, Prf, and Rx-mediated cell death, while the NRC3b variants failed to rescue cell death mediated by these sensors. Interestingly, most NRC3c variants were able to function with Prf and Rx but not Rpi-blb2 (Fig 1D). The accumulations of all the natural variants were detectable, and none of them showed strong auto-activity in inducing cell death when expressed alone (Figs 1E and S3). These results suggested that NRC3 homologs have evolved to be functionally divergent. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Rpi-blb2 signals through NRC3a but not NRC3b or NRC3c. (A) Cell death assays of Rpi-blb2 with different NRCs. Rpi-blb2 and AVRblb2 were co-expressed with indicated NRCs cloned from tomato and N. benthamiana in nrc2/3/4_KO N. benthamiana. Cell death intensity and phenotypes were recorded at 6 dpi. The line in the boxplots represents the medium, the box edges represent the 25th and 75th percentiles, and the whiskers extend to the most extreme data points no more than 1.5x of the interquartile range. Statistical differences between the negative control (EV) and tested groups were examined by paired Wilcoxon signed rank test (* = p < 0.0001). (B) Assays of Rpi-blb2-mediated cell death rescued by NRC variants. Rpi-blb2 and AVRblb2 were co-expressed with NRCs as indicated in both WT or nrc2/3/4_KO N. benthamiana. (C) Phylogenetic analysis of NRC3 natural variants identified from tomato, tobacco potato, pepper, and eggplant. Sequence alignment of the NB-ARC domain was used to generate the phylogenetic tree using the Maximum likelihood method with 1000 bootstrap tests. SlNRC1 and SlNRC2 were selected as outgroups. The scale bars indicate the evolutionary distance in amino acid substitution per site. The orange, green, and blue boxes indicate allelic groups A, B, and C, respectively. (D) Cell death assays of different sensor NLRs with NRC3 natural variants. The cloned NRC3 natural variants were co-expressed with Rpi-blb2/AVRblb2, Pto/AvrPto, or Rx/CP in nrc2/3/4_KO N. benthamiana. (E) Protein accumulation of NRC3 natural variants. NRC3 natural variants were transiently expressed in WT N. benthamiana. The proteins were extracted from leaf samples at 2 dpi and the NRC3 protein accumulations were detected by α-myc antibody. SimplyBlue SafeStain-staining of Rubisco was used as the loading control. The dot plots represent cell death intensity quantified by UVP ChemStudio PLUS at 6 dpi. The line in the boxplots represents the medium, the box edges represent the 25th and 75th percentiles, and the whiskers extend to the most extreme data points no more than 1.5x of the interquartile range. Statistical differences between the negative control (EV) and tested groups were examined by paired Wilcoxon signed rank test (* = p<0.05, ** = p < 0.0001). https://doi.org/10.1371/journal.pgen.1011402.g001

Ancestral reconstructions reveal subfunctionalization of NRC3c towards loss of compatibility with Rpi-blb2 To determine which molecular events contributed to the functional divergence of NRC3, we performed functional assays of reconstructed ancestral NRC3 variants. We extracted 324 non-redundant nucleotide sequences of the NRCX, NRC1, NRC2, and NRC3 clades from 124 solanaceous genomes, and then used FastML to reconstruct the ancestral NRC sequences (S4A Fig). We synthesized the full-length of five ancestral NRC3 variants reconstructed from the FastML, including N4, N95, N89, N88, and N3 that represent the ancestral state of NRC3a, NRC3b, NRC3c, NRC3b/c, and before the divergence of the three allelic groups (Figs 2A and S5). We then tested the degree to which these ancestral variants can rescue Rpi-blb2, Prf (Pto), and Rx-mediated cell death. We found that while N3, N4, and N88 rescue Rpi-blb2-mediated cell death, the ancestral variants N95 (NRC3b) and N89 (NRC3c) show no or low activities in rescuing Rpi-blb2-mediated cell death (Fig 2B). While most of these ancestral variants (N3, N4, N88, and N89) rescued Prf (Pto) and Rx-mediated cell death, N95 was the only variant that failed to rescue any cell death tested (Fig 2B). The accumulations of all of these ancestral variants were detectable with low or no auto-activity (S6 Fig). We introduced a D to V mutation into the MHD motif of N88, N95 and NRC3b variants, and found that, while N88DV induced very strong cell death, none of the N95DV and NRC3bDV variants induced cell death in N. benthamiana, suggesting that this group of NRC3 has nonfunctionalized during the evolutionary process (Fig 2C). These results indicate that NRC3 in the ancestral species likely functions together with Rpi-blb2/Prf/Rx, whereas the NRC3 variants that evolved in Nicotiana species acquired mutations leading to nonfunctionalization (NRC3b) or subfunctionalization (NRC3c), losing their ability to work together with Rpi-blb2. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Subfunctionalization contributes to the evolution of NRC3c. (A) Phylogenetic tree of NRC3 natural variants. Orange, green, and blue boxes represent allelic groups A, B, and C, respectively. Red dots indicate the nodes of reconstructed ancestral NRC3 variants. (B) Cell death assays of ancestral NRC3 variants. The ancestral variants were co-expressed with Rpi-blb2/AVRblb2, Pto/AvrPto, or Rx/CP in nrc2/3/4_KO N. benthamiana. (C) Cell death analysis of NRC3b_DV, N88_DV and N95_DV. The NRC3_DV variants carry a D to V mutation in the MHD motif. These variants were expressed alone in WT N. benthamiana. The dot plots represent cell death intensity quantified by UVP ChemStudio PLUS at 6 dpi. The line in the boxplots represents the medium, the box edges represent the 25th and 75th percentiles, and the whiskers extend to the most extreme data points no more than 1.5x of the interquartile range. Statistical differences between the negative control (EV) and tested groups were examined by paired Wilcoxon signed rank test (* = p < 0.0001). https://doi.org/10.1371/journal.pgen.1011402.g002 To further test this hypothesis, we synthesized NRC3 of Petunia inflata (PinNRC3) which is sister to the three NRC3 allelic groups mentioned above (Fig 1C). We noticed that the PinNRC3 from the genome database contains an indel of 9 amino acids between the CC and NB-ARC domains (S7A Fig). Therefore, we manually curated the sequence by inserting 9 amino acids from SlNRC3a into the indel of PinNRC3, and found that this manually curated PinNRC3 variant is able to rescue all three cell death phenotypes tested (S7B Fig). Taken together, these results support the hypothesis that the ancestral NRC3 can function with a broader collection of sensor NLRs, while NRC3c variants subfunctionalized to work with a smaller subset of sensor NLRs.

Two K to N mutations in the NB-ARC and LRR domains play critical roles in NRC3 subfunctionalization Next, we looked into the polymorphism of these 6 positions in the NRC3 natural variants mentioned above (Figs 1C and 3G). We found that all the NRC3 variants from allelic group A possess PKKTHK, and all the variants from allelic group B and the outgroup PinNRC3 possess PKKTRK (Fig 4A). Interestingly, sequences from allelic group C showed higher diversity, with NbNRC3c being the most diverse variant (Fig 4A). We introduced these different polymorphisms into the NbNRC3c background and tested the ability of these variants to rescue Prf and Rpi-blb2 cell death in nrc2/3/4_KO N. benthamiana. While all these variants rescued Prf-mediated cell death, NRC3 variants with PKKTHK or PKKTRK, but not PKNTRN or PTNTRN, were able to fully rescue Rpi-blb2-mediated cell death (Figs 4A and S16). Consistent with the results from the polymorphisms found in the natural variants, the major differences between N88 (PKKTRK) and N89 (PKNTRN) were also the two K to N mutations (Figs 3G and 4B). These findings indicate that the two mutations converting K to N are likely the most crucial changes during the subfunctionalization process. To further test this hypothesis, we introduce the two K to N mutations into the ancestral variants N88 (N88NN) or the N to K mutations into the ancestral variants N89 (N89KK). We found that N88NN showed reduced activity and N89KK showed increased activity compared to their ancestral states respectively (Figs 4B and S17). These results support the finding that six amino acid residues contribute to the compatibility of NRC3 variants to Rpi-blb2, with two K to N changes playing major roles in the NRC3 subfunctionalization process. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Two K to N mutations in the NB-ARC and LRR domains play major roles in NRC3 subfuntionalization. (A) Left panel, the polymorphisms in the NRC3 natural variants at the 6 positions identified. Right panel, cell death assay testing these polymorphisms in NbNRC3c background. Introducing PKKTHK or PKKTRK enabled it to function with Rpi-blb2. (B) Left panel, the polymorphisms in the ancestral NRC3 variants at the 6 positions identified. Right panel, cell death assay testing two lysine-asparagine changes in both N88 and N89 backgrounds. Swapping two K to N in N88/N89 changed the compatibility to Rpi-blb2. The dot plots in (A) and (B) represent cell death intensity quantified by UVP ChemStudio PLUS at 6 dpi. The line in the boxplots represents the medium, the box edges represent the 25th and 75th percentiles, and the whiskers extend to the most extreme data points no more than 1.5x of the interquartile range. Statistical differences were examined using Dunn’s test (p < 0.05) (C) The entropy analysis of natural NRC3 variants. The protein sequences of NRC3s were aligned using MAFFT and the Shannon entropy was calculated. The positions of the two K to N changes were highlighted in green. (D) dN-dS calculation of natural NRC3 variants by using SLAC analysis. The positions of K to N changes were highlighted in green. The two residues were under neutral selection based on FEL analysis. https://doi.org/10.1371/journal.pgen.1011402.g004 To understand the polymorphisms of these two positions across NRC3 alleles, we performed entropy analysis using the protein sequence alignment of the natural NRC3 variants. We found that these positions both had entropy values of 0.637 and did not stand out as being among the most conserved or diversified positions (Fig 4C and S4 Dataset). We then calculated the dN-dS value using SLAC (Single-Likelihood Ancestor Counting) and found that the residue at position 221 showed a slightly higher dN than dS value, while the residue at position 832 showed a lower dN value than dS value (Fig 4D and S5 Dataset). Despite this, the FEL (Fixed Effects Likelihood) analysis indicated that both positions are under neutral selection (Fig 4D and S5 Dataset). These results suggest that variations in these amino acids among different NRC3 allelic groups, leading to subfunctionalization, are more likely the outcome of random genetic drift rather than the consequence of strong selection.

Sensor-helper compatibility is determined by multiple protein surfaces To understand the spatial arrangement of the residues involved in sensor-helper compatibility on the NRC3 structure, we performed predictions using AlphaFold2 and fitted the structure model onto the recently published NRC2 resting homodimer (S18 Fig) [19]. We then highlighted the residues involved in sensor-helper compatibility on the predicted resting NbNRC3c homodimer complex (Fig 5A). We found that N832(K), located near the end of the LRR, is in proximity to S202(P) and T203(K) in the NB-ARC domain, suggesting they may be on the same exposed surface of the resting complex of NRC3 (Fig 5B). Both of the residues I642(T) and C824(H) are on the concave surface of the LRR domain, facing a cavity in between the LRR and the NB-ARC domain (Fig 5C). Interestingly, the residue N221(K) is located in the region between the two NbNRC3c protomers, corresponding to interface 1a described in the NRC2 homodimer (Fig 5D) [19]. This residue is also positioned next to the cavity in between the LRR and the NB-ARC domain (Fig 5C). PPT PowerPoint slide

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TIFF original image Download: Fig 5. Multiple protein surface contribue to the sensor-helper compatibility determination. (A) Structure of NbNRCc homodimer shown in two orthogonal views. The NB-ARC domains of two protomers are shown in light blue and the LRR domains are shown in pink. The 6 residues involved in sensor-helper compatibility are highlighted in yellow, green, and red. (B) Details of the exposed surface between S202(P)/T203(K) and N832(K). (C) Details of the cavity in between the NB-ARC domain and the LRR domain. This cavity is surrounded by I642(T), C824(H), and N221(K). (D) Details of the interface between two NbNRC3c protomers where the N221(K) is located. The interfaces are highlighted based on the resting NbNRC2 homodimer. (E) Cell death assays of NRC3 variants carrying identified residues from SlNRC3 in only two of the surfaces. The residues on the exposed surface, on the concave surface of the LRR domain, and on the interface between NRC3 protomers are highlighted in yellow, green, and red, respectively. Cell death assays were performed by co-expressing chimeric NRC3 variants with Rpi-blb2/AVRblb2 in nrc2/3/4_KO N. benthamiana. The dot plots represent cell death intensity quantified by UVP ChemStudio PLUS at 6 dpi. The line in the boxplots represents the medium, the box edges represent the 25th and 75th percentiles, and the whiskers extend to the most extreme data points no more than 1.5x of the interquartile range. Statistical differences were examined using Dunn’s test (p < 0.05). https://doi.org/10.1371/journal.pgen.1011402.g005 To further investigate whether the three surfaces collectively contribute to sensor-helper compatibility, we generated variants containing residues from SlNRC3 in only two of the surfaces using NNN as the background (Fig 5E). Compared to the NN PKK N THK variant, all these three new variants (NN PKN N THK , NN PKK N ICK , and NN STK N THN ) only showed partial activities in rescuing Rpi-blb2-mediated cell death (Fig 5E). All of these variants rescued Prf-mediated cell death, with no auto-activity, and accumulated to a similar level (S19 Fig). These results suggested that the three surfaces on NRC3 contribute to the sensor-helper compatibility collectively.

Steady-state interactions between sensor and helper NLRs do not reflect their compatibility To further dissect the molecular mechanisms of sensor-helper compatibility, we tested the interactions between NRC3 variants with the sensor NLR Rpi-blb2. We focused on two variants, NNN (NbNRC3c) and NN PKK N THK , as these two variants differ from each other by only six amino acids but display robust differences in their compatibility with Rpi-blb2 (Fig 3H). We co-expressed Rpi-blb2 with NNN or NN PKK N THK with or without AVRblb2 and then performed co-immunoprecipitation analyses. When we pulled down Rpi-blb2, we detected very weak signals from NNN and NN PKK N THK regardless of whether AVRblb2 was present or not (S20A Fig). Similarly, when we pulled down the two NRC3 variants, we detected weak signals from Rpi-blb2 (S20B Fig). These results suggest that steady-state interactions between sensor and helper NLRs of the NRC superclade detected using co-IP experiments do not reflect their compatibility.

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