- Some members of the RLP family are known to be involved in basal. - We found that the two classes differ in abundance on both transcriptome and proteome level, physical clustering in the genome and putative interaction partners. - However, the classes do not differ in the genetic di versity of their individual members in accessible pan-genome data.. - Most known are the intracellular receptor genes of the NLR family (nucleotide-binding domain and leucine-rich repeat containing receptor family). - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - An- other study investigating sequence polymorphisms in NLRs from a single tomato species found that NLRs ex- perience different selective pressures dependent on the geographical location of the population [6]. - These stud- ies therefore highlight that defence-associated gene fam- ilies appear to be highly diverse but do not allow comparisons between defence- and development- associated genes in the same gene family.. - These domains can facilitate bind- ing and recognition of the corresponding ligands or en- able interaction with other proteins to maintain or finetune signalling [7]. - Members of the RLP family have been shown to be involved in both develop- mental and defence mechanisms, making them ideally suited to investigate whether functional differences lead to differences in rates of evolution.. - Of the 57 annotated RLPs in Arabidopsis thaliana, 2 RLPs are experimentally validated to be associated with developmental functions (RLP10/CLV2, RLP17/TMM), and 6 with defence functions (RLP . - Fritz-Laylin et al. - PDO1/RLP51 is the underlying gene of the snc2-1D locus (for suppressor of npr1, con- stitutive 2-1D), a semidominant gain-of-function Arabi- dopsis thaliana mutant with dwarf morphology and constitutively activated defense responses including high salicylic acid and PATHOGENESIS-RELATED (PR) genes levels [17]. - There is some debate as to whether the separation of the recognised molecules (PAMPs vs.. - RLP32 recognizes the structural fold of the bacterial translation initiation factor − 1 (Inf-1) present in all proteobacteria [31] and RLP42/RBPG1 detects sev- eral endopolygalacturonases from Botrytis cinerea and Aspergillus niger [28]. - Finally, RLP3 is the causal gene of the quantitative resistance locus RFO2 in Arabidopsis. - As these 6 RLPs (RLP and 42) play important roles in the defence against various pathogens we will refer to them as VDRs (validated defence RLPs) in the remainder of this manuscript.. - thali- ana reference genome, the gene expression atlas, an Arabidopsis transcriptome and proteome database, the sequencing data from the 1001 Arabidopsis genome pro- ject as well as a copy number variant atlas to gain a dee- per understanding of the function and putative role of the RLP family in Arabidopsis. - Defence- and development-associated RLPs cluster differently in the phylogenetic tree. - We redid the phylo- genetic analysis as previously performed by Wang et al.. - Our tree resembles the phylogeny by Wang et al., [9] with high support values for most internal branches (Fig. - The PDOs, except RLP46, are all on the basal branches of the phylogenetic tree, whereas the defence- associated RLPs are more scattered across the tree and also populate the larger non-basal part. - This is in line with previous publications where already a higher num- ber of RLPs was predicted to be associated with defence and where it was further shown that 47 out of the ana- lyzed 57 RLPs cluster within superclades where at least one member was defence-associated [10].. - Based on the findings above, we hypothesized that RLPs on the upper branches of the tree are more likely to beassociated with defence. - To expand the annotation data of the RLPs, we used the Genevestigator software [35]. - Thirty-five RLPs showed upregulated gene expression patterns after treat- ment with pathogens in at least one of the different pathogen infection datasets, whereas 17 RLPs showed no changes in expression after pathogen treatment in any of the examined data sets. - 1 Phylogenetic tree of the conserved C3-F domain of RLPs. - °AtRLP25 is not up or down regulated at all and AtRLP8 was not present in the used datasets. - whereas only one of the previously annotated PDOs show changes after infection (RLP46). - 1, yellow box) yet almost no difference in the expression levels after pathogen infection of the RLPs clustering in the lower, basal branches (Fig. - The only four exceptions are the two VDRsd, RLP1 and RLP3, as well as RLP21 which are all basal in the tree, but show a gene upregulation after infections. - RLP25 shows no changes in expression in any of the examined datasets and RLP8 was missing from the data.. - When combined, these data suggest that the upper part of the phylogenetic tree most likely contains defence-associated RLPs that are all derived from more ancestral, putative developmental-related RLPs. - In the remainder of this manuscript we will therefore refer to the upper part of the phylogeny as prRLPs (pathogen-re- sponsive RLPs) and the lower part as bRLPs (basal RLPs).. - Similar to Wang et al. - All of the VDRs possess the conserved stretch of nega- tively charged amino acids (Aspartate (D) and Glutamate. - We expanded these analyses and investigated the presence of these motifs in the complete prRLP set and the non- pathogen responsive bRLP set and found that with only one exception all pathogen-responsive RLPs contain both motifs, whereas one or in some cases even both motifs ap- pear to be absent in the bRLP set (Fig. - For many RLPs expression data was only available for very few of the analyzed tissues. - Closer inspection shows that the fraction of RLPs with a detectable transcript differs significantly between prRLPs and bRLPs, with a lower fraction detected in the prRLPs:. - When calculating hierarch- ical clustering on protein abundance, we also observed a clustering of the pr- and bRLPs, although it is less obvi- ous than on the expression level (Fig. - Similar to the transcriptome data, the proteome data show significant differences between the fraction of RLPs present in the pathogen-responsive fraction (prRLPs. - It has been hypothesized that the physical location of defence-associated genes, like those in the NLRs and RLP families, allows for more rapid evolution and re- combination and that as such, these gene families evolved in clusters on the genome. - This show that the frac- tion of clustered RLPs is higher in the pathogen- responsive RLPs (27 clustered, 7 singletons) than in the basal (7 clustered, 13 singletons. - 2 Alignment of the extracellular juxtamembrane, transmembrane and cytoplasmic region of the RLPs. - Most of the prRLPs (boxed in yellow) have both motifs required for interaction with SOBIR1, the negatively charged amino acid stretch in the eJM and the GxxxG-motif in the TM, except RLP28 lacking both motifs and RLP46 having only two Gs. - Most of the bRLPs (boxed in blue) lack the GxxxG-motif, except RLP44 and RLP57, but the latter lack a dominant negatively-charged amino acid stretch in the eJM. - Color coded in magenta are the Glycines (G) in the TM and in cyan the Aspartates (D) and Glutamates (E) in the eJM. - of the prRLPs and of the bRLPS have no such SNPs. - Looking specifically in the clustered and non- clustered RLPs revealed no difference in the absence or presence of SNPs between RLPs which are encoded as a single gene or those in pairs or larger clusters (40 % [8/. - our surprise, the nucleotide diversity measured as π/site is significantly larger in the bRLPs (Fig. - Lastly, we found no significant differences in the ratio of non-. - This separation is further confirmed by analysis per- formed by Fritz-Laylin et al. - Two protein- interacting motifs are required for RLP-SOBIR1 inter- action, which is a negatively-charged stretch of amino-acids in the extracellular juxtamembrane re- gion and a GxxxG-motif in the transmembrane do- main [30]. - Alignment of these regions showed that in most of the prRLPs both of the motifs are present, whereas in the bRLPs they are less common or com- pletely absent, suggesting that interaction with SOBIR1 is defence-specific.. - We analysed the transcriptomic and proteomic expres- sion profiles of the RLPs in 30 different samples repre- senting different tissues and different development stages [37] and compared the prRLPs with the bRLPs.. - Our data showed that all known RLPs reported to function as PRRs show similar patterns in transcriptome and proteome data, especially regarding the presence of the respective protein in an uninduced state. - All of the predicted RLPs belong to the prRLPs and the protein is present in an uninduced state. - This might be because some of the bRLPs have dual roles (like CLV2) [11], or because the cluster of bRLPs also contains some defence-associated RLPs. - Yet the stark differences in the amount of poly- morphisms in some prRLPs as well as some bRLPs might indicate specific roles for these more diverse RLPs.. - Recently, such intra- genic recombinations have been shown to play a major role in the maintenance of stable polymorphisms in an. - infestans in the wild potato species Solanum ameri- canum [48], as well as in the RLP locus Hcr9, conferring resistance against the fungal pathogen Cladosporium ful- vum in wild tomato [49].. - Overall, by combining several public resources, we en- hance current knowledge of the RLP gene family in Ara- bidopsis. - Further research on the identification of the role of RLPs in various cellular processes will help to better understand the observed differences within the RLPs and prove our suggested hypothetical grouping into prRLPs and bRLPs. - The phylogenetic tree of the Arabidopsis’ RLPs was made using the conserved C3-F domain, as previously reported by Wang et al. - The phylogenetic tree of the RLPs from tomato and Arabidopsis was made by Kang and Yeom [42] using the amino acid sequences of the C3-F domains using PhyMl.. - The resulting multiple sequence alignments can be found in the supplementary materials.. - De- tailed experimental procedures on data generation and normalization can be found in the accompanying paper [37]. - Missing data were imputed around the mean using random numbers drawn from the lower part of the normal distri- bution with the standard settings for width (0.3) and downward shift (1.8) in the Perseus software (v Width: Defines the width of the Gaussian distribu- tion relative to the standard deviation of measured values. - For each of the two data sets (normalized and imputed transcriptomic and proteomic data) we calculated the Pearson correlations between the normalised TPM values for the transcriptome and normalised iBAQ values for the proteome in R, using cor, followed by clustering using hclust (method = complete for proteome and ward.d2 for transcriptome) and plotting as.dendo- gram. - The normalized data and scripts for clustering can be found in the supplementary materials.. - The genomic clustering is based on the analysis done by Tör et al. - To test whether the observed number of bRLPs and prRLPs in clusters differed from expected values, we used the χ 2 -test (chisq.test) as imple- mented in the R stats package.. - The genomic coordi- nates of the CNVs were extracted from the supplemen- tary data and converted to bed format. - All above mentioned RLP an- notations and scripts for the calculations can be found in the supplementary information online.. - Scripts can be found in the supplementary information online.. - We would like to thank all the authors of the used resources for publishing open access and sharing their data with the community, without it this whole work wouldn ’ t have been possible. - Intermediate data files, such as multiple sequence alignments, mentioned in the manuscript as well as the R script used for the analysis of the transcriptome, proteome, 1001 genome diversity as well as those used to calculate the correlations between the expected classification and specific features (clustering, upregulation, etc. - Stam R, Nosenko T, Hörger AC, Stephan W, Seidel M, Kuhn JMM, et al. - The de Novo Reference Genome and Transcriptome Assemblies of the Wild Tomato Species Solanum chilense Highlights Birth and Death of NLR Genes Between Tomato Species. - Van de Weyer A-L, Monteiro F, Furzer OJ, Nishimura MT, Cevik V, Witek K, et al. - Wang G, Ellendorff U, Kemp B, Mansfield JW, Forsyth A, Mitchell K, et al. - Phylogenomic Analysis of the Receptor-Like Proteins of Rice and Arabidopsis. - Pan L, Lv S, Yang N, Lv Y, Liu Z, Wu J, et al. - Functional Analyses of the CLAVATA2-Like Proteins and Their Domains That Contribute to CLAVATA2 Specificity. - Holzwart E, Huerta AI, Glöckner N, Gómez BG, Wanke F, Augustin S, et al.. - BRI1 controls vascular cell fate in the Arabidopsis root through RLP44 and phytosulfokine signaling. - Zhang Y, Yang Y, Fang B, Gannon P, Ding P, Li X, et al. - Jehle AK, Lipschis M, Albert M, Fallahzadeh-Mamaghani V, Fürst U, Mueller K, et al. - Perception of the novel MAMP eMax from different Xanthomonas species requires the Arabidopsis receptor-like protein ReMAX and the receptor kinase SOBIR. - Albert I, Böhm H, Albert M, Feiler CE, Imkampe J, Wallmeroth N, et al. - Zhang W, Fraiture M, Kolb D, Löffelhardt B, Desaki Y, Boutrot FF, et al.. - Zhang L, Kars I, Essenstam B, Liebrand TWH, Wagemakers L, Elberse J, et al.. - Shared and distinct functions of the pseudokinase CORYNE (CRN) in shoot and root stem cell maintenance of Arabidopsis. - Lin G, Zhang L, Han Z, Yang X, Liu W, Li E, et al. - Berardini TZ, Reiser L, Li D, Mezheritsky Y, Muller R, Strait E, et al. - Mass- spectrometry-based draft of the Arabidopsis proteome. - AthCNV: A Map of DNA Copy Number Variations in the Arabidopsis Genome. - Genome-wide Identification, Classification, and Expression Analysis of the Receptor-Like Protein Family in Tomato. - Witek K, Lin X, Karki HS, Jupe F, Witek AI, Steuernagel B, et al. - Population studies of the wild tomato species Solanum chilense reveal geographically structured major gene-mediated pathogen resistance
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