Genomics of the NB genes in cassava: from gene expression to integrative biology

Genomics of the NB genes in cassava: from gene expression to integrative biology | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genomics of the NB genes in cassava: from gene expression to integrative biology Margelly Andrea Bastidas Pardo, David Santiago Padilla-Fino, Maria Alejandra Rojo, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7624510/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Proteins containing the NB (Nucleotide Binding) domain are intracellular receptors or co-receptors that recognize proteins from microorganisms directly or indirectly. Plant genomes contain a variable number of NB genes, but the function of most of them remains unknown. Cassava is a crucial staple crop, and given its drought-tolerant nature, it is a promising crop for addressing climate change. Here, we describe the manual curation of 262 NB genes present in the cassava genome. The corresponding proteins were classified according to the presence of additional domains such as TIR (Toll Interleukin-1 Receptor) and/or LRR (Leucine Rich Repeat). The gene expression of these genes was evaluated using several transcriptomic experiments available in databases. Our analysis revealed that most NB genes are expressed at low or very low levels, with around 20% of them showing high expression values. The differential expression analysis detected 26 differentially expressed NB genes of which nine correspond to NB proteins lacking one or two motifs present in the NB domain. An integrative approach that took into account gene expression, polymorphisms level, and co-expression network metrics demonstrated that some NB proteins lacking certain motifs play important roles in the network structure, despite their low expression. On the other hand, the same analysis highlights the importance of complete NB proteins that showed higher levels of fold change. Several NB genes represent excellent candidates for further functional validation studies and/or breeding programs. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Plant immunity relies on a complex system of molecular receptors able to detect proteins or other pathogen-derived molecules (Ngou et al., 2022 ). The first kind of immunity receptors are called Pattern Recognition Receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs). PRRs are receptors containing an extracellular Leucine-Rich Repeats (LRR) domain, which can be accompanied by a kinase cytoplasmic domain (Boutrot and Zipfel, 2019). The second large group of plant immunity receptors are the NB (nucleotide-binding)-LRR proteins, which recognize pathogen effectors (Yue et al., 2012 ). Depending on the presence of additional domains in their N-termini, NB-LRR proteins can be classified as Toll/interleukin-1 receptor (TIR)-NB-LRR (TNL) and non-TNL types. Usually, non-TNL proteins have a coiled-coil (CC) domain (Deyoung et al., 2006; Contreras et al., 2023a ). The NB domain includes key motifs essential for ATP and GTP binding and hydrolysis, such as the P-loop, Kinase-2, and Kinase-3 motifs. These motifs enable conformational changes necessary for protein transition between inactive and active states, a critical feature for signal transduction (Lukasik and Takken, 2009 ). Both PRRs and NB-LRR receptors, once recognizing pathogen molecules, trigger a signal pathway activating plant immunity (Ngou et al., 2022 ). Cassava ( Manihot esculenta ) is a staple crop critical to the food security of around one billion people in tropical regions worldwide (Parmar et al., 2017 ). Its starchy roots provide a primary source of carbohydrates for developing nations. Cassava's ability to grow well in poor soils and under adverse conditions, such as drought and nutrient limitations, makes it an invaluable resource for communities with limited agricultural inputs and in the context of climate change (Cock and Connor, 2021 ). Despite its importance, cassava productivity is threatened by various biotic stresses, including bacterial, fungal, and viral diseases (Mc Callum et al., 2017). The most significant bacterial disease is cassava bacterial blight, caused by Xanthomonas phaseoli pv. manihotis (Xpm), which can reduce yields by 20–100%, depending on infection severity (Zarate-Chaves et al., 2021). Enhancing cassava's resistance through genetic improvement, particularly by targeting NB proteins that play critical roles in immune responses, offers a sustainable solution. A comprehensive bioinformatics approach was conducted to elucidate the genomic structure and response of NB genes in cassava. From an initial set of 565 automatically annotated NB genes, manual curation yielded 262 genes. Structural analysis identified different combinations of motifs in the NB domain. Gene expression analysis demonstrated low and basal expression of most NB genes, although induction was observed in some cases. An integrative approach including gene expression, co-expression network, and SNPs (Single Nucleotide Polymorphisms) provided a holistic view of NB genes and their importance during cassava responses to different stimuli. Materials and Methods Identification of NB Genes, Manual Annotation, and SNPs The NB Pfam accession number (PF00931) was used to query the cassava genome v8 available at Phytozome ( https://phytozome-next.jgi.doe.gov/ ). Nucleotide and corresponding protein sequences were downloaded and manually curated. Blastp analysis was conducted to confirm the presence and similarity with other NB-containing proteins. Additionally, the MOTIF database ( https://www.genome.jp/tools/motif/ ) was employed to confirm the presence of NB and delimit the presence of NB domain motifs. The amino acids corresponding only to the NB domain were extracted and a phylogenetic tree was constructed using ClustalX from the multiple alignment using the Neighborhood Joining Method (Larkin et al., 2007 ). Single Nucleotide Polymorphisms (SNPs) were identified in cassava varieties using Genotyping-by-Sequencing (GBS) methods in previously generated data (Mora et al., in press ). The presence and frequency of polymorphisms for each NB gene were obtained exploring the database manually. Expression Profiling Using Public RNA-seq Data Data from the NCBI database were queried based on criteria such as biological relevance, data accessibility, statistical robustness (number of samples and replicates), and quality of experimental design (RNA-seq or high-throughput sequencing techniques). Six experiments focused on cassava's interaction with microorganisms, including four specifics to Xpm, one involving mycorrhiza and one with whitefly. Data from three other experiments investigating cassava's responses to abiotic stresses were also retrieved. Raw RNA sequencing (RNA-Seq) data in FASTQ format from five projects (PRJNA257332, PRJNA688032, PRJNA491633, PRJNA396755, PRJNA911587) (Table 1 ) stored in the Sequence Read Archive (SRA) platform of NCBI were utilized. FastQC was used for initial sequence quality assessment, and Trimmomatic version 0.39 was used for data cleaning with default parameters. Filtered reads were aligned to the Manihot esculenta version 8 reference genome using STAR version 2.7.9a. For the remaining four datasets (GSE82279, GSE234712, GSE53369, GSE141125) (Table 1 ), count tables were available directly from NCBI's Gene Expression Omnibus. SAMtools was used to organize generated BAM files. Gene expression data were obtained using HTSeq-count version 0.13.5, configured to process position-based reads and identify genes based on a GTF annotation file. Table 1 Result summary and description of the transcriptomic cassava data sets. a Indicates the number of differentially expressed NB genes detected when all genes were used for the analysis. Experiment Description # of NB very high expression (%) # of NB very low expression (%) # NB genes expressed # of NB DE (all) 1 # NB DE (NB) 2 A PRJNA257332 Control : Cassava leaves collected at 8, 24, and 50 hours after treatment with MgCl₂ Treatments : cassava leaves inoculated with i ) Xanthomonas euvesicatoria containing the TAL20 form Xpm668 ; ii ) X. euvesicatoria and iii ) Xanthomonas phaseoli strain 668. Samples collected at 8, 24, and 50 hours post-inoculation. 33 (12,7) 59 (22,7) 260 1 8 B PRJNA396755 Control : Cassava stems non inoculated and collected at 1, 3, and 5 days after the experiment Treatments cassava stem inoculated with MgCl₂ or with Xpm681 and collected at 1, 3, and 5 days after inoculation 70 (26,7) 48 (18,3) 262 0 0 C PRJNA491633 Control different tissues collected at 0, 3, and 24 hours after the experiment. Treatment : PEG-induced water stress. tissues collected at 0, 3, and 24 hours post-treatment 26(10,0) 52 (20,1) 259 3 0 D PRJNA688032 Control : Cassava leaves non inoculated and collected at 50 hours after treatment with MgCl₂ Treatment : cassava leaves inoculated with two Xpm strains (XamUA1061 and XamUA681) collected at 50 hours post-infection 29 (11,8) 48 (19,5) 246 1 1 E PRJNA911587 Control : Cassava plants non inoculated Treatment : cassava plants inoculated with Rhizophagus and collected at 3 and 6 weeks after infection 89 (34,1) 56 (21,5) 261 1 3 F GSE82279 Control: FEC and OES Treatments lateral bud, leaf, fibrous root, midvein, petiole, RAM, SAM, and stem; storage root c . 23 (16,4) 18 (12,9) 140 11 11 G GSE234712 Control : Cassava plants non infested Treatment : cassava plants infested with whitefly samples collected at 1, 3, 5, and 10 days post-inoculation 16 (10,1) 29 (18,2) 159 0 0 H GSE53369 Control : Cassava stems inoculated with a non-pathogenic strain (ORST4) and collected at 0, 3, and 5 days after inoculation Treatments cassava stems inoculated with ORST4 strain modified with the effector TALE1Xam collected at 1, 3, and 5 days after inoculation 15 (16,7) 12 (13,3) 90 3 3 I GSE141125 Control : Cassava plants cultivar KU50 cultivated in Hanoi and collected at 1, 2, 3 and 4 months after planting? Treatment : Cassava plants cultivar KU50 cultivated in VolverKan and collected at 1, 2, 3 and 4 months after planting 26 (17,1) 19 (12,5) 152 5 7 1 Indicates the result obtained when only NB genes were used for the analysis. 2 For the differential gene expression analyses FEC and OES were considered as control Differential Expression Analysis Each experiment was analyzed separately, and outliers and irrelevant genes were filtered out. Expression patterns of NB genes were visualized, and expression levels were categorized into high, medium, and low based on percentiles 33.3 and 66.6. For differential expression analysis, data were separated into control and stressed conditions, and DESeq2 analysis was performed using R packages. Integrative Biology For co-expression network analysis, expression profile data across all nine datasets were centered and transformed with rlog. An adjacency matrix was generated from the Pearson correlation similarity gene-gene matrix, and gene pairs passing the similarity threshold based on Elo et al. (2007) were included in the network. Topological metrics (degree, centrality, HubCentrality, and Betweenness) were calculated for NB genes in the network using the igraph package (Gábor and Nepusz, 2006). All information was integrated into a multivariate data table and used for Principal Component Analysis to characterize cassava's NB coding genes. Results Initial Identification and Structural Analysis The initial analysis based on the presence of the NB domain (PF00931) in the cassava genome available in Phytozome retrieved 565 candidate genes. However, rigorous curation and manual annotation reduced the number to 262. Based on the presence of additional domains, the NB protein-coding genes were grouped into five classes (Supplementary Table 1). The largest class contained 196 genes and corresponded to genes containing the NB and LRR domains (NL), followed by a class containing an additional TIR domain (TNL) with 34 genes. Twenty-eight genes coded for proteins containing only the NB domain (N), and four genes corresponded to proteins having the TIR and NB domains but lacking the LRR (TN). A detailed inspection of the motifs in the NB allowed us to identify the presence/absence of some of the three classical motifs conforming the NB: P-loop, kinase-2, and kinase-3. Different combinations were detected (Supplementary Table 1). The majority of them (143 genes) had the three motifs. However, 36 genes had only the motifs P-loop and kinase-2, while 31 genes had the P-loop + Kinase-3 motifs. Thirteen genes lacked the P-loop, and finally, 10 genes contained only the P-loop motif. Polymorphisms and Evolutionary Relationship Taking advantage of the partial genome sequences obtained by Genotyping By Sequence (GBS) for a panel of 182 cassava cultivars (Mora et al., in press ), a search for SNPs was performed on the 262 NB coding genes. In total, 3,282 SNPs were identified. 18% of the genes coding for NB proteins showed no SNPs. The genes with fewer than 10 SNPs were the most abundant (Fig. 1 ). However, high frequencies of polymorphisms were observed, with 4.8% of the genes showing more than 70 SNPs (Fig. 1 ). The gene Manes_02G209475v8 had the highest level of polymorphism with 116 SNPs. (Supplementary table 1). To determine the evolutionary relationship among the cassava NB genes, a phylogenetic tree based on the alignment of the NB sequence for the 262 genes was constructed. Clearly, two main groups of genes were observed, separating the two largest classes of genes, the NL and the TNL. The N class was distributed along the NLs (Fig. 2 ). Transcriptome Analysis With the aim of studying NB expression, RNA-seq data available in the NCBI Gene Expression Omnibus (GEO) database were retrieved, selecting nine different experiments (Table 1 ). Initially, we sought to evaluate the expression of the 262 NB genes in different organs or tissues (dataset F_GSE82279). Around half of the 262 NB genes (122; 47%) showed zero expression in any of the organs. On the other hand, 140 genes were expressed (53%), of which 23 (16%) and 18 genes (13%) were very highly and very lowly expressed, respectively. Notably, although with minor changes, the expression profile for the storage root was the most different. Taken together, 26 NB genes were differentially expressed: 17 o which belong to the category with all three motifs, two of them to the P-loop and Kinase2 category, six to the Kinase2 and Kinase3 category and only one to the Kinase3 category. No differentially expressed genes were detected for the other categories (P-loop and Kinase3, P-loop, Kinase2). These results demonstrate the constitutive expression of some NB genes in different cassava organs and differences related to the motifs present in the gene (Supplementary Table 1). The expression of NB genes was examined in response to drought (C_PRJNA491633); in this case, only three genes were not expressed. However, just 26 genes (10%) showed strong expression. The leaves and roots collected from plants treated or not with PEG showed a very similar pattern of NB expression. Four particular NB genes (Manes_11G130000v8, Manes_13G002200v8, Manes_10G112000v8, and Manes_05G169600v8) showed higher expression in roots. When the total set of genes was considered for differential expression analysis, three NB genes showed statistically different expression. Although when only the NB genes were considered for the analysis, no NB genes were detected. The NB gene expression was evaluated during biotic responses, including cassava- Xanthomonas interaction (A_PRJNA25733, B_PRJNA396755, D_PRJNA688032, H_GSE53369), cassava- Rhizophagus interaction (E_PRJNA25733), and cassava infected with whitefly (G_GSE234712). The first dataset of cassava interacting with Xanthomonas (A_PRJNA25733) corresponds to plants infected with Xpm or with the bacteria Xanthomonas euvesicatoria (Xe) containing the TALE20 from Xpm. This study aimed to evaluate the effect of the TALE protein on cassava gene expression during the disease. In this case, almost the entire set of NB genes showed at least a low count of reads (260). Among the expressed genes, 33 (13%) showed a very high expression level, and 59 (23%) were very lowly expressed. The expression profile was almost identical between plants inoculated with Xpm or Xe and non-inoculated plants, although at 8 hpi, a group of NB genes were more expressed, regardless of whether the plants were inoculated or not. The differential expression analysis allowed the detection of eight NB genes differentially expressed when only the NB genes were considered. However, only one showed differential expression when the complete dataset of genes was considered in the analysis. In the second dataset (B_PRJNA396755), cassava plants from the cultivar 60444 were inoculated with Xpm 681, representing a susceptible reaction. Similar to the previous dataset, all the NB genes were expressed, although in this case, the genes highly expressed were slightly more numerous (70 genes, representing 27%). The genes with very low expression were 48 (18%). In this case, none of the NB genes showed statistically different expression. In another experiment, the same cassava cultivar was inoculated with two Xpm strains (UA681 and UA1061) to evaluate the effect of two strains containing different TALEs on cassava responses, and this constituted the third dataset (D_PRJNA688032). Sixteen NB genes were not expressed in any of the conditions studied. Notably, in this group, most of them corresponded to those lacking the kinase-3 motif. The number of NB genes expressed at a major level was 29 (12%). The gene expression profile was similar in controls and inoculated plants, although the differential gene expression analysis allowed the detection of one NB gene expressed differentially between control and inoculated plants. Finally, in the fourth dataset (H_GSE53369), plants were inoculated with a non-pathogenic Xpm strain (ORST4) and the same strain but containing a TALE protein. Curiously, in this case, the majority of the NB genes were not expressed (172, 65%), and from the 90 remaining NB genes, 15 of them showed high expression. The expression profiles were also very similar between conditions; however, at 5 days post-inoculation, regardless of the Xpm strain, a small group of around five NB genes were more expressed, of which three showed statistically different expression. During the cassava interaction with Rhizophagus (E_PRJNA25733), all the NB genes except one were expressed, and particularly, most of them (89 genes; 34%) showed relatively high expression. The visual transcriptomic heatmap was almost identical between plants in the presence of Rhizophagus or not; however, one or three NB genes were differentially expressed, depending on whether the analysis was conducted considering all genes or only the NB genes, respectively. The last experiment in the biotic response evaluated the gene expression profile during the infection of cassava plants with whitefly (G_GSE234712). 159 NB genes were expressed in at least one of the conditions, and only 16 (10%) of them were expressed at significant levels. Minor differences were observed in the expression profiles between non-infested and infested plants at different times, which was corroborated by the differential analysis that did not detect any NB gene expressed differentially. Finally, the expression of NB genes was explored using data obtained from two different regions to compare the environmental effect on flowering. In this case, 152 NB genes were expressed in at least one condition. Among the genes expressed, 15 (17%) and 12 (13%) showed high and very low expression, respectively. In this case, it was not possible to visually identify a particular condition favouring or not the induction or repression of a specific group of NB genes. However, when comparing expression between the two regions, five or seven NB genes were expressed differentially, depending on whether the complete list of genes or only the NB genes were used to conduct the analysis. Co-expression Network Once the NB gene expression was evaluated, understanding not only the magnitude of their expression but also how their expression is coordinated with each other within a broader network was crucial. A gene co-expression network was built, where nodes represent genes and edges (links) indicate co-expression interactions between them, reflecting how their expression is coordinated. The co-expression network exhibited modularity, with clusters of genes co-regulated under specific stress conditions. The analysis identified several hub genes with high centrality and extensive co-expression relationships. The genes Manes_02G209475v8, Manes_02G201200v8, Manes_10G023100v8, Manes_02G202959v8, Manes_02G221720v8, and Manes_07G063100v8 emerged as central nodes within the network, with high connectivity values. This indicates that these genes are strongly connected with other genes in the network and that their co-expression may reflect their joint participation in specific biological processes rather than direct control over the expression of other genes (Topological measures can be found in Supplementary Table 1). This high connectivity suggests that these genes could be fundamental for the structural and functional organization of the network in response to internal or external stimuli. These genes were predominantly members of the NL class. Genes with high betweenness scores were also noted as critical for connecting various functional modules. Integrated Information To integrate all the information generated for the NB genes (gene expression, NB classification, polymorphisms, network parameters, etc.), a Principal Component Analysis (PCA) was performed on all variables (Supplementary Table 1). This multivariate analysis provided insights into the major sources of variability and relationships among the analyzed genes. The PCA identified several principal components, with the first three explaining a significant proportion of the variance in the dataset (~ 40%). PC1 captured 20.5% of the total variability and was strongly associated with network centrality metrics, such as degree and hub centrality. PC2 accounted for 12.2% of the variance and was mainly associated with the number of SNPs (Fig. 4 ). The SNPs were strongly correlated with the network structure but also showed a correlation with the fold change in NB gene expression in experiments A, C, and E on one side and with experiments B and G on the other. The PCA biplot allowed identifying that the majority of NB genes were grouped in the upper right part of the graph (Fig. 4 ), indicating that they have no major changes in terms of gene expression and do not contribute significantly to the network. However, some "atypical" genes have extreme characteristics in terms of the evaluated variables. For example, genes 1(Manes_10G133686), 2 (Manes_10G133698), 4 (Manes_10G128106), and 6 (Manes_10G112000) showed extreme values in terms of network centrality parameters and belong to the P-loop-kinase-2 class. On the other hand, genes having all the motifs present in the NB, such as genes 12 (Manes_13G002100), 163 (Manes_03G060801), 26 (Manes_09G025400), and 52 (Manes_11G156500), were localized at the extremes of the axis associated with the fold change, indicating differential gene expression for particular experiments. Discussion In this work, we obtained 262 fully manually annotated genes coding for NB from an initial number of 562 genes retrieved from the cassava genome database. These 262 NB genes were classified into five groups depending on the presence/absence of additional domains. The NB domain for the majority of them contained the three classical motifs. The transcriptomics profile of these genes in response to different biotic and abiotic stimuli and in different tissues allowed us to confirm their low and constitutive expression, although some genes showed slight induction. The expression analysis identified some differentially expressed genes, particularly during the cassava- Xanthomonas interaction. The co-expression network allowed the identification of hub and central genes. Finally, an integrative approach provided a comprehensive association between expression, structure, and variability of the NB genes. The number of NB genes varies drastically between species, with numbers as low as around 50 in melon or papaya to more than 1000 in apple or wheat (Contreras et al., 2023), and there is no correlation between the number of NB genes and genome size (Hussain et al., 2024 ). In this study, we identified 262 NB genes, which are within the range observed for other plant species. From these 262 genes, 146 correspond to the NL class and 34 to the TNL class. Soto et al. (2014) reported 177 and 28, while Lozano et al. ( 2015 ) described 117 and 29, respectively. Although the numbers are not identical, they are quite similar. Differences can be partially explained by the genome version employed or to the algorithms and/or curation carried out. In this work, meticulous manual annotation and curation were conducted; consequently, the genes identified here can be considered the most confident. Interestingly, the NB genes present in the cassava genome contain different combinations of presence/absence or typical motifs conforming to this domain. Nearly half (143) of the NB contained the three motifs; however, the remaining NB (119) had different combinations of two motifs, and some had only one. The NB lacking complete motifs have been considered non-functional or pseudogenes and have been previously reported in cassava (Lozano et al., 2015 ). The function of these "incomplete" NB is unknown, although it has been proposed that they can serve as a reservoir to generate diversity of genes with new specificities (Meyers et al., 2003 ). For some of these NB genes, the heatmaps showed low or no expression, suggesting they could be non-functional. However, 14 NB genes lacking one or two motifs were differentially expressed in some experiments, suggesting they can participate in responses to stimuli. These truncated or motif-lacking NB can interact with complete functional NB to immunomodulate them, as reported for NRG1C (Wu et al., 2022 ). Interestingly, the integrative model showed that several NB containing only the P-loop and kinase-2 motifs proved to be important in giving structure to the network, although their genes were not differentially expressed or their fold change was lower. Thus, these neglected kinds of genes should be considered important for modulating and orchestrating an efficient immune response. The evaluation of NB transcriptomics in cassava during different conditions showed that, although most experiments expressed NB genes, only between 10% and 34% of them were expressed at relatively high levels. Lozano et al. ( 2015 ) reported around 77% of NB genes were constitutively expressed in cassava leaves. Historically, the expression of NB genes has been considered low and constitutive, given that the proteins should be present even before infection to detect pathogens. From this perspective, NB genes are not necessarily induced to be functional. In most conditions studied here, NB genes were expressed at low levels. However, in some cases, they were induced, notably in response to Xpm (experiment B) and Rhizophagus (experiment E). Among the 27 differentially expressed NB genes, 13 belonged to the category containing the three motifs, which are probably functional in activating responses. In particular, genes such as 26 (Manes_09G025400), 34 (Manes_09G024872), and 163 (Manes_03G060801), were localized at the periphery of the biplot, indicating an important contribution of their differential expression to the plant response. Several recent reports have shown the induction of particular NB genes in response to pathogens (Zhang et al., 2015; Yu et al., 2021; Kan et al., 2024; Shi et al., 2021 ; Liu et al., 2019 ). Taken together, these results and previous ones reinforce the importance of NB in mounting a response and that they can be regulated at the transcriptional level. Previous transcriptomic studies during cassava interactions with pathogens such as the virus CBSV (Maruthi et al., 2014 ) and Xpm (Muñoz Bodnar et al., 2014) have not detected differentially expressed NB genes. In this work, we conducted two different strategies to identify DEGs. In the first approach, the information of all annotated genes was included, and in the second one, the analysis was conducted only considering the NB genes. Generally, more differentially expressed NB genes were detected when the second approach was used. This highlights once again that the low expression level of NB genes can mask their identification as DEGs. By combining different analyses, it was possible to gain a deep understanding of the structure, expression, diversity, and concerted action of the different NB proteins in cassava. The integrative information provided new putative functions for NB lacking complete motifs and underscored the importance of transcriptional regulation of NB genes in responding to different stimuli, notably biotic responses. From this analysis, it was possible to identify NB genes that can be considered strong candidates for further functional validation and potential employment in breeding programs. Declarations Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors Author Contribution CEL and LLK conceptualized the study, analyzed the data and wrote the manuscript. MAB, DSP and MAR collected, analyzed the data and prepared the figures. Acknowledgements The authors would like to thank the students of the course in Statistical Genomic of the Statistics Department of Universidad Nacional de Colombia – sede Bogotá Juan Felipe Avella Duque and Diana Carolina Briñez Perales for their participation in the first transcriptomic data analysis used for this work and all members of the Manihot Biotec research group from Universidad Nacional de Colombia – sede Bogotá for their input during the development of the study. Data Availability The data supporting the findings of this study are already available at NCBI (PRJNA257332, PRJNA688032, PRJNA491633, PRJNA396755, PRJNA911587 and GSE82279, GSE234712, GSE53369, GSE141125) References Boutrot F, Zipfel C (2017) Function, discovery, and exploitation of plant pattern recognition receptors for broad-spectrum disease resistance. Annu Rev Phytopathol 55:257–286 Cock J, Connor D (2021) Cassava. In: Sadras VO, Calderini DF (eds) Crop Physiology Case Histories for Major Crops. Academic, Amsterdam, pp 588–633 Contreras MP, Lüdke D, Pai H, Toghani A, Kamoun S (2023a) NLR receptors in plant immunity: making sense of the alphabet soup. EMBO Rep 24:e57495 Deyoung BJ, Innes RW (2006) Plant NBS–LRR proteins in pathogen sensing and host defense. Nat Immunol 7:1243–1249 Hussain A, Khan AA, Aslam MQ et al (2024) Comparative analysis, diversification, and functional validation of plant nucleotide-binding site domain genes. 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New Phytol 193:1049–1063 Zárate-Chaves CA, de la Gómez D, Verdier V, López CE, Bernal A, Szurek B (2021) Cassava diseases caused by Xanthomonas phaseoli pv. manihotis and Xanthomonas cassavae. Mol Plant Pathol 22:1520–1537 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7624510","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":544508618,"identity":"db85eb5f-9625-4054-9e53-329a2b3f84ae","order_by":0,"name":"Margelly Andrea Bastidas Pardo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Margelly","middleName":"Andrea Bastidas","lastName":"Pardo","suffix":""},{"id":544508619,"identity":"acba1d09-0f19-409e-b62e-6f3939c2fbb6","order_by":1,"name":"David Santiago Padilla-Fino","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"Santiago","lastName":"Padilla-Fino","suffix":""},{"id":544508620,"identity":"cfdf560d-b40f-454c-aca5-15faf90e830f","order_by":2,"name":"Maria Alejandra Rojo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Alejandra","lastName":"Rojo","suffix":""},{"id":544508621,"identity":"120f6910-eccd-4bd8-a264-3c5b78bdcc65","order_by":3,"name":"Liliana López-Kleine","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Liliana","middleName":"","lastName":"López-Kleine","suffix":""},{"id":544508622,"identity":"74cad394-eefd-4052-84fd-d094dde89c6e","order_by":4,"name":"Camilo Ernesto Lopez 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19:05:49","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":81710,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7624510/v1/bcbd4d2a57e6ac66f3cf69db.html"},{"id":96662936,"identity":"610f0b6f-7344-4d9d-be61-4a31a2b8c30e","added_by":"auto","created_at":"2025-11-24 19:05:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14905,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistogram of the number of SNPs (Npolym) per NB gene.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7624510/v1/a7abdc5ab967fb91f29b56ff.png"},{"id":96709729,"identity":"3e0ca105-da02-4159-bd2d-1ecd836c324f","added_by":"auto","created_at":"2025-11-25 10:09:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":624845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of the 262 NB sequences. T: TIR (Toll interleukin 1 receptor) domain, N: NB (nucleotide binding) domain, L: LRR (leucin rich repeat) domain.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7624510/v1/02d3dd71e55ee45ea2572124.png"},{"id":96662950,"identity":"09275630-a782-4d69-a849-158a39df4998","added_by":"auto","created_at":"2025-11-24 19:05:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":397531,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of each reflecting the expression level of each of the nine experiments analyzed. See Table 1 for a description of each experiment.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7624510/v1/163256419b459fc8b1993d4a.png"},{"id":96662939,"identity":"b931e855-3ed7-4f2b-b4ab-4805666cec2f","added_by":"auto","created_at":"2025-11-24 19:05:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":216883,"visible":true,"origin":"","legend":"\u003cp\u003eBiplot of the Principal component analysis integrating all variables obtained for the NB genes.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7624510/v1/0533d7509d9ca2d81d5628a9.png"},{"id":98428273,"identity":"723eb673-16f5-40c3-bab7-222b049dceaf","added_by":"auto","created_at":"2025-12-17 16:41:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1942064,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7624510/v1/40aaa35b-2988-4e9b-a6cd-649fc114777f.pdf"}],"financialInterests":"","formattedTitle":"Genomics of the NB genes in cassava: from gene expression to integrative biology","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant immunity relies on a complex system of molecular receptors able to detect proteins or other pathogen-derived molecules (Ngou et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The first kind of immunity receptors are called Pattern Recognition Receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs). PRRs are receptors containing an extracellular Leucine-Rich Repeats (LRR) domain, which can be accompanied by a kinase cytoplasmic domain (Boutrot and Zipfel, 2019). The second large group of plant immunity receptors are the NB (nucleotide-binding)-LRR proteins, which recognize pathogen effectors (Yue et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Depending on the presence of additional domains in their N-termini, NB-LRR proteins can be classified as Toll/interleukin-1 receptor (TIR)-NB-LRR (TNL) and non-TNL types. Usually, non-TNL proteins have a coiled-coil (CC) domain (Deyoung et al., 2006; Contreras et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe NB domain includes key motifs essential for ATP and GTP binding and hydrolysis, such as the P-loop, Kinase-2, and Kinase-3 motifs. These motifs enable conformational changes necessary for protein transition between inactive and active states, a critical feature for signal transduction (Lukasik and Takken, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Both PRRs and NB-LRR receptors, once recognizing pathogen molecules, trigger a signal pathway activating plant immunity (Ngou et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCassava (\u003cem\u003eManihot esculenta\u003c/em\u003e) is a staple crop critical to the food security of around one billion people in tropical regions worldwide (Parmar et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Its starchy roots provide a primary source of carbohydrates for developing nations. Cassava's ability to grow well in poor soils and under adverse conditions, such as drought and nutrient limitations, makes it an invaluable resource for communities with limited agricultural inputs and in the context of climate change (Cock and Connor, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite its importance, cassava productivity is threatened by various biotic stresses, including bacterial, fungal, and viral diseases (Mc Callum et al., 2017). The most significant bacterial disease is cassava bacterial blight, caused by \u003cem\u003eXanthomonas phaseoli\u003c/em\u003e pv. \u003cem\u003emanihotis\u003c/em\u003e (Xpm), which can reduce yields by 20\u0026ndash;100%, depending on infection severity (Zarate-Chaves et al., 2021). Enhancing cassava's resistance through genetic improvement, particularly by targeting NB proteins that play critical roles in immune responses, offers a sustainable solution.\u003c/p\u003e\u003cp\u003eA comprehensive bioinformatics approach was conducted to elucidate the genomic structure and response of NB genes in cassava. From an initial set of 565 automatically annotated NB genes, manual curation yielded 262 genes. Structural analysis identified different combinations of motifs in the NB domain. Gene expression analysis demonstrated low and basal expression of most NB genes, although induction was observed in some cases. An integrative approach including gene expression, co-expression network, and SNPs (Single Nucleotide Polymorphisms) provided a holistic view of NB genes and their importance during cassava responses to different stimuli.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of NB Genes, Manual Annotation, and SNPs\u003c/h2\u003e\u003cp\u003eThe NB Pfam accession number (PF00931) was used to query the cassava genome v8 available at Phytozome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Nucleotide and corresponding protein sequences were downloaded and manually curated. Blastp analysis was conducted to confirm the presence and similarity with other NB-containing proteins. Additionally, the MOTIF database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/tools/motif/\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/tools/motif/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to confirm the presence of NB and delimit the presence of NB domain motifs. The amino acids corresponding only to the NB domain were extracted and a phylogenetic tree was constructed using ClustalX from the multiple alignment using the Neighborhood Joining Method (Larkin et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Single Nucleotide Polymorphisms (SNPs) were identified in cassava varieties using Genotyping-by-Sequencing (GBS) methods in previously generated data (Mora et al., \u003cem\u003ein press\u003c/em\u003e). The presence and frequency of polymorphisms for each NB gene were obtained exploring the database manually.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExpression Profiling Using Public RNA-seq Data\u003c/h3\u003e\n\u003cp\u003eData from the NCBI database were queried based on criteria such as biological relevance, data accessibility, statistical robustness (number of samples and replicates), and quality of experimental design (RNA-seq or high-throughput sequencing techniques). Six experiments focused on cassava's interaction with microorganisms, including four specifics to Xpm, one involving mycorrhiza and one with whitefly. Data from three other experiments investigating cassava's responses to abiotic stresses were also retrieved.\u003c/p\u003e\u003cp\u003eRaw RNA sequencing (RNA-Seq) data in FASTQ format from five projects (PRJNA257332, PRJNA688032, PRJNA491633, PRJNA396755, PRJNA911587) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) stored in the Sequence Read Archive (SRA) platform of NCBI were utilized. FastQC was used for initial sequence quality assessment, and Trimmomatic version 0.39 was used for data cleaning with default parameters. Filtered reads were aligned to the Manihot esculenta version 8 reference genome using STAR version 2.7.9a. For the remaining four datasets (GSE82279, GSE234712, GSE53369, GSE141125) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), count tables were available directly from NCBI's Gene Expression Omnibus. SAMtools was used to organize generated BAM files. Gene expression data were obtained using HTSeq-count version 0.13.5, configured to process position-based reads and identify genes based on a GTF annotation file.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResult summary and description of the transcriptomic cassava data sets. a Indicates the number of differentially expressed NB genes detected when all genes were used for the analysis.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperiment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDescription\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e# of \u003cem\u003eNB\u003c/em\u003e very high expression (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e# of \u003cem\u003eNB\u003c/em\u003e very low expression (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e# \u003cem\u003eNB\u003c/em\u003e genes expressed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e# \u0026nbsp; of NB DE (all)\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e# NB DE (NB)\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA PRJNA257332\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e: Cassava leaves collected at 8, 24, and 50 hours after treatment with MgCl₂\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatments\u003c/b\u003e: cassava leaves inoculated with \u003cem\u003ei\u003c/em\u003e) Xanthomonas euvesicatoria containing the TAL20 form Xpm668 ; \u003cem\u003eii\u003c/em\u003e) X. euvesicatoria and \u003cem\u003eiii\u003c/em\u003e) Xanthomonas phaseoli strain 668. Samples collected at 8, 24, and 50 hours post-inoculation.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33 (12,7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e59 (22,7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e260\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB\u003c/p\u003e\u003cp\u003ePRJNA396755\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e: Cassava stems non inoculated and collected at 1, 3, and 5 days after the experiment\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatments\u003c/b\u003e cassava stem inoculated with MgCl₂ or with Xpm681 and collected at 1, 3, and 5 days after inoculation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e70 (26,7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e48 (18,3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e262\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC\u003c/p\u003e\u003cp\u003ePRJNA491633\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e different tissues collected at 0, 3, and 24 hours after the experiment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatment\u003c/b\u003e: PEG-induced water stress. tissues collected at 0, 3, and 24 hours post-treatment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26(10,0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e52 (20,1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e259\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD\u003c/p\u003e\u003cp\u003ePRJNA688032\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e: Cassava leaves non inoculated and collected at 50 hours after treatment with MgCl₂\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatment\u003c/b\u003e: cassava leaves inoculated with two Xpm strains (XamUA1061 and XamUA681) collected at 50 hours post-infection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29 (11,8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e48 (19,5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e246\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eE\u003c/p\u003e\u003cp\u003ePRJNA911587\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e: Cassava plants non inoculated\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatment\u003c/b\u003e: cassava plants inoculated with Rhizophagus and collected at 3 and 6 weeks after infection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e89 (34,1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56 (21,5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e261\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF\u003c/p\u003e\u003cp\u003eGSE82279\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl: FEC and OES\u003c/p\u003e\u003cp\u003eTreatments lateral bud, leaf, fibrous root, midvein, petiole, RAM, SAM, and stem; storage root\u003csup\u003ec\u003c/sup\u003e.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23 (16,4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18 (12,9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e140\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eG\u003c/p\u003e\u003cp\u003eGSE234712\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e: Cassava plants non infested\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatment\u003c/b\u003e: cassava plants infested with whitefly\u003c/p\u003e\u003cp\u003esamples collected at 1, 3, 5, and 10 days post-inoculation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16 (10,1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e29 (18,2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e159\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH\u003c/p\u003e\u003cp\u003eGSE53369\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e: Cassava stems inoculated with a non-pathogenic strain (ORST4) and collected at 0, 3, and 5 days after inoculation\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatments\u003c/b\u003e cassava stems inoculated with ORST4 strain modified with the effector TALE1Xam collected at 1, 3, and 5 days after inoculation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15 (16,7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12 (13,3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI\u003c/p\u003e\u003cp\u003eGSE141125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e: Cassava plants cultivar KU50 cultivated in Hanoi and collected at 1, 2, 3 and 4 months after planting?\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatment\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eCassava plants cultivar KU50 cultivated in VolverKan and collected at 1, 2, 3 and 4 months after planting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26 (17,1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19 (12,5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e152\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Indicates the result obtained when only NB genes were used for the analysis.\u003c/p\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e For the differential gene expression analyses FEC and OES were considered as control\u003c/p\u003e\n\u003ch3\u003eDifferential Expression Analysis\u003c/h3\u003e\n\u003cp\u003eEach experiment was analyzed separately, and outliers and irrelevant genes were filtered out. Expression patterns of NB genes were visualized, and expression levels were categorized into high, medium, and low based on percentiles 33.3 and 66.6. For differential expression analysis, data were separated into control and stressed conditions, and DESeq2 analysis was performed using R packages.\u003c/p\u003e\n\u003ch3\u003eIntegrative Biology\u003c/h3\u003e\n\u003cp\u003eFor co-expression network analysis, expression profile data across all nine datasets were centered and transformed with rlog. An adjacency matrix was generated from the Pearson correlation similarity gene-gene matrix, and gene pairs passing the similarity threshold based on Elo et al. (2007) were included in the network.\u003c/p\u003e\u003cp\u003eTopological metrics (degree, centrality, HubCentrality, and Betweenness) were calculated for NB genes in the network using the igraph package (G\u0026aacute;bor and Nepusz, 2006). All information was integrated into a multivariate data table and used for Principal Component Analysis to characterize cassava's NB coding genes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eInitial Identification and Structural Analysis\u003c/h2\u003e\u003cp\u003eThe initial analysis based on the presence of the NB domain (PF00931) in the cassava genome available in Phytozome retrieved 565 candidate genes. However, rigorous curation and manual annotation reduced the number to 262. Based on the presence of additional domains, the NB protein-coding genes were grouped into five classes (Supplementary Table\u0026nbsp;1). The largest class contained 196 genes and corresponded to genes containing the NB and LRR domains (NL), followed by a class containing an additional TIR domain (TNL) with 34 genes. Twenty-eight genes coded for proteins containing only the NB domain (N), and four genes corresponded to proteins having the TIR and NB domains but lacking the LRR (TN). A detailed inspection of the motifs in the NB allowed us to identify the presence/absence of some of the three classical motifs conforming the NB: P-loop, kinase-2, and kinase-3. Different combinations were detected (Supplementary Table\u0026nbsp;1). The majority of them (143 genes) had the three motifs. However, 36 genes had only the motifs P-loop and kinase-2, while 31 genes had the P-loop\u0026thinsp;+\u0026thinsp;Kinase-3 motifs. Thirteen genes lacked the P-loop, and finally, 10 genes contained only the P-loop motif.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePolymorphisms and Evolutionary Relationship\u003c/h3\u003e\n\u003cp\u003eTaking advantage of the partial genome sequences obtained by Genotyping By Sequence (GBS) for a panel of 182 cassava cultivars (Mora et al., \u003cem\u003ein press\u003c/em\u003e), a search for SNPs was performed on the 262 NB coding genes. In total, 3,282 SNPs were identified. 18% of the genes coding for NB proteins showed no SNPs. The genes with fewer than 10 SNPs were the most abundant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, high frequencies of polymorphisms were observed, with 4.8% of the genes showing more than 70 SNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The gene Manes_02G209475v8 had the highest level of polymorphism with 116 SNPs. (Supplementary table 1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine the evolutionary relationship among the cassava NB genes, a phylogenetic tree based on the alignment of the NB sequence for the 262 genes was constructed. Clearly, two main groups of genes were observed, separating the two largest classes of genes, the NL and the TNL. The N class was distributed along the NLs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eTranscriptome Analysis\u003c/h3\u003e\n\u003cp\u003eWith the aim of studying NB expression, RNA-seq data available in the NCBI Gene Expression Omnibus (GEO) database were retrieved, selecting nine different experiments (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInitially, we sought to evaluate the expression of the 262 NB genes in different organs or tissues (dataset F_GSE82279). Around half of the 262 NB genes (122; 47%) showed zero expression in any of the organs. On the other hand, 140 genes were expressed (53%), of which 23 (16%) and 18 genes (13%) were very highly and very lowly expressed, respectively. Notably, although with minor changes, the expression profile for the storage root was the most different. Taken together, 26 NB genes were differentially expressed: 17 o which belong to the category with all three motifs, two of them to the P-loop and Kinase2 category, six to the Kinase2 and Kinase3 category and only one to the Kinase3 category. No differentially expressed genes were detected for the other categories (P-loop and Kinase3, P-loop, Kinase2). These results demonstrate the constitutive expression of some NB genes in different cassava organs and differences related to the motifs present in the gene (Supplementary Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eThe expression of NB genes was examined in response to drought (C_PRJNA491633); in this case, only three genes were not expressed. However, just 26 genes (10%) showed strong expression. The leaves and roots collected from plants treated or not with PEG showed a very similar pattern of NB expression. Four particular NB genes (Manes_11G130000v8, Manes_13G002200v8, Manes_10G112000v8, and Manes_05G169600v8) showed higher expression in roots. When the total set of genes was considered for differential expression analysis, three NB genes showed statistically different expression. Although when only the NB genes were considered for the analysis, no NB genes were detected.\u003c/p\u003e\u003cp\u003eThe NB gene expression was evaluated during biotic responses, including cassava-\u003cem\u003eXanthomonas\u003c/em\u003e interaction (A_PRJNA25733, B_PRJNA396755, D_PRJNA688032, H_GSE53369), cassava-\u003cem\u003eRhizophagus\u003c/em\u003e interaction (E_PRJNA25733), and cassava infected with whitefly (G_GSE234712). The first dataset of cassava interacting with \u003cem\u003eXanthomonas\u003c/em\u003e (A_PRJNA25733) corresponds to plants infected with Xpm or with the bacteria \u003cem\u003eXanthomonas euvesicatoria\u003c/em\u003e (Xe) containing the TALE20 from Xpm. This study aimed to evaluate the effect of the TALE protein on cassava gene expression during the disease. In this case, almost the entire set of NB genes showed at least a low count of reads (260). Among the expressed genes, 33 (13%) showed a very high expression level, and 59 (23%) were very lowly expressed. The expression profile was almost identical between plants inoculated with Xpm or Xe and non-inoculated plants, although at 8 hpi, a group of NB genes were more expressed, regardless of whether the plants were inoculated or not. The differential expression analysis allowed the detection of eight NB genes differentially expressed when only the NB genes were considered. However, only one showed differential expression when the complete dataset of genes was considered in the analysis.\u003c/p\u003e\u003cp\u003eIn the second dataset (B_PRJNA396755), cassava plants from the cultivar 60444 were inoculated with Xpm 681, representing a susceptible reaction. Similar to the previous dataset, all the NB genes were expressed, although in this case, the genes highly expressed were slightly more numerous (70 genes, representing 27%). The genes with very low expression were 48 (18%). In this case, none of the NB genes showed statistically different expression.\u003c/p\u003e\u003cp\u003eIn another experiment, the same cassava cultivar was inoculated with two Xpm strains (UA681 and UA1061) to evaluate the effect of two strains containing different TALEs on cassava responses, and this constituted the third dataset (D_PRJNA688032). Sixteen NB genes were not expressed in any of the conditions studied. Notably, in this group, most of them corresponded to those lacking the kinase-3 motif. The number of NB genes expressed at a major level was 29 (12%). The gene expression profile was similar in controls and inoculated plants, although the differential gene expression analysis allowed the detection of one NB gene expressed differentially between control and inoculated plants.\u003c/p\u003e\u003cp\u003eFinally, in the fourth dataset (H_GSE53369), plants were inoculated with a non-pathogenic Xpm strain (ORST4) and the same strain but containing a TALE protein. Curiously, in this case, the majority of the NB genes were not expressed (172, 65%), and from the 90 remaining NB genes, 15 of them showed high expression. The expression profiles were also very similar between conditions; however, at 5 days post-inoculation, regardless of the Xpm strain, a small group of around five NB genes were more expressed, of which three showed statistically different expression.\u003c/p\u003e\u003cp\u003eDuring the cassava interaction with \u003cem\u003eRhizophagus\u003c/em\u003e (E_PRJNA25733), all the NB genes except one were expressed, and particularly, most of them (89 genes; 34%) showed relatively high expression. The visual transcriptomic heatmap was almost identical between plants in the presence of \u003cem\u003eRhizophagus\u003c/em\u003e or not; however, one or three NB genes were differentially expressed, depending on whether the analysis was conducted considering all genes or only the NB genes, respectively.\u003c/p\u003e\u003cp\u003eThe last experiment in the biotic response evaluated the gene expression profile during the infection of cassava plants with whitefly (G_GSE234712). 159 NB genes were expressed in at least one of the conditions, and only 16 (10%) of them were expressed at significant levels. Minor differences were observed in the expression profiles between non-infested and infested plants at different times, which was corroborated by the differential analysis that did not detect any NB gene expressed differentially.\u003c/p\u003e\u003cp\u003eFinally, the expression of NB genes was explored using data obtained from two different regions to compare the environmental effect on flowering. In this case, 152 NB genes were expressed in at least one condition. Among the genes expressed, 15 (17%) and 12 (13%) showed high and very low expression, respectively. In this case, it was not possible to visually identify a particular condition favouring or not the induction or repression of a specific group of NB genes. However, when comparing expression between the two regions, five or seven NB genes were expressed differentially, depending on whether the complete list of genes or only the NB genes were used to conduct the analysis.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCo-expression Network\u003c/h2\u003e\u003cp\u003eOnce the NB gene expression was evaluated, understanding not only the magnitude of their expression but also how their expression is coordinated with each other within a broader network was crucial. A gene co-expression network was built, where nodes represent genes and edges (links) indicate co-expression interactions between them, reflecting how their expression is coordinated. The co-expression network exhibited modularity, with clusters of genes co-regulated under specific stress conditions.\u003c/p\u003e\u003cp\u003eThe analysis identified several hub genes with high centrality and extensive co-expression relationships. The genes Manes_02G209475v8, Manes_02G201200v8, Manes_10G023100v8, Manes_02G202959v8, Manes_02G221720v8, and Manes_07G063100v8 emerged as central nodes within the network, with high connectivity values. This indicates that these genes are strongly connected with other genes in the network and that their co-expression may reflect their joint participation in specific biological processes rather than direct control over the expression of other genes (Topological measures can be found in Supplementary Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eThis high connectivity suggests that these genes could be fundamental for the structural and functional organization of the network in response to internal or external stimuli. These genes were predominantly members of the NL class. Genes with high betweenness scores were also noted as critical for connecting various functional modules.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eIntegrated Information\u003c/h2\u003e\u003cp\u003eTo integrate all the information generated for the NB genes (gene expression, NB classification, polymorphisms, network parameters, etc.), a Principal Component Analysis (PCA) was performed on all variables (Supplementary Table\u0026nbsp;1). This multivariate analysis provided insights into the major sources of variability and relationships among the analyzed genes.\u003c/p\u003e\u003cp\u003eThe PCA identified several principal components, with the first three explaining a significant proportion of the variance in the dataset (~\u0026thinsp;40%). PC1 captured 20.5% of the total variability and was strongly associated with network centrality metrics, such as degree and hub centrality. PC2 accounted for 12.2% of the variance and was mainly associated with the number of SNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe SNPs were strongly correlated with the network structure but also showed a correlation with the fold change in NB gene expression in experiments A, C, and E on one side and with experiments B and G on the other. The PCA biplot allowed identifying that the majority of NB genes were grouped in the upper right part of the graph (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), indicating that they have no major changes in terms of gene expression and do not contribute significantly to the network.\u003c/p\u003e\u003cp\u003eHowever, some \"atypical\" genes have extreme characteristics in terms of the evaluated variables. For example, genes 1(Manes_10G133686), 2 (Manes_10G133698), 4 (Manes_10G128106), and 6 (Manes_10G112000) showed extreme values in terms of network centrality parameters and belong to the P-loop-kinase-2 class. On the other hand, genes having all the motifs present in the NB, such as genes 12 (Manes_13G002100), 163 (Manes_03G060801), 26 (Manes_09G025400), and 52 (Manes_11G156500), were localized at the extremes of the axis associated with the fold change, indicating differential gene expression for particular experiments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, we obtained 262 fully manually annotated genes coding for NB from an initial number of 562 genes retrieved from the cassava genome database. These 262 NB genes were classified into five groups depending on the presence/absence of additional domains. The NB domain for the majority of them contained the three classical motifs.\u003c/p\u003e\u003cp\u003eThe transcriptomics profile of these genes in response to different biotic and abiotic stimuli and in different tissues allowed us to confirm their low and constitutive expression, although some genes showed slight induction. The expression analysis identified some differentially expressed genes, particularly during the cassava-\u003cem\u003eXanthomonas\u003c/em\u003e interaction. The co-expression network allowed the identification of hub and central genes. Finally, an integrative approach provided a comprehensive association between expression, structure, and variability of the NB genes.\u003c/p\u003e\u003cp\u003eThe number of NB genes varies drastically between species, with numbers as low as around 50 in melon or papaya to more than 1000 in apple or wheat (Contreras et al., 2023), and there is no correlation between the number of NB genes and genome size (Hussain et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, we identified 262 NB genes, which are within the range observed for other plant species.\u003c/p\u003e\u003cp\u003eFrom these 262 genes, 146 correspond to the NL class and 34 to the TNL class. Soto et al. (2014) reported 177 and 28, while Lozano et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) described 117 and 29, respectively. Although the numbers are not identical, they are quite similar. Differences can be partially explained by the genome version employed or to the algorithms and/or curation carried out. In this work, meticulous manual annotation and curation were conducted; consequently, the genes identified here can be considered the most confident.\u003c/p\u003e\u003cp\u003eInterestingly, the NB genes present in the cassava genome contain different combinations of presence/absence or typical motifs conforming to this domain. Nearly half (143) of the NB contained the three motifs; however, the remaining NB (119) had different combinations of two motifs, and some had only one. The NB lacking complete motifs have been considered non-functional or pseudogenes and have been previously reported in cassava (Lozano et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe function of these \"incomplete\" NB is unknown, although it has been proposed that they can serve as a reservoir to generate diversity of genes with new specificities (Meyers et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). For some of these NB genes, the heatmaps showed low or no expression, suggesting they could be non-functional. However, 14 NB genes lacking one or two motifs were differentially expressed in some experiments, suggesting they can participate in responses to stimuli.\u003c/p\u003e\u003cp\u003eThese truncated or motif-lacking NB can interact with complete functional NB to immunomodulate them, as reported for NRG1C (Wu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Interestingly, the integrative model showed that several NB containing only the P-loop and kinase-2 motifs proved to be important in giving structure to the network, although their genes were not differentially expressed or their fold change was lower.\u003c/p\u003e\u003cp\u003eThus, these neglected kinds of genes should be considered important for modulating and orchestrating an efficient immune response. The evaluation of NB transcriptomics in cassava during different conditions showed that, although most experiments expressed NB genes, only between 10% and 34% of them were expressed at relatively high levels.\u003c/p\u003e\u003cp\u003eLozano et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) reported around 77% of NB genes were constitutively expressed in cassava leaves. Historically, the expression of NB genes has been considered low and constitutive, given that the proteins should be present even before infection to detect pathogens. From this perspective, NB genes are not necessarily induced to be functional.\u003c/p\u003e\u003cp\u003eIn most conditions studied here, NB genes were expressed at low levels. However, in some cases, they were induced, notably in response to Xpm (experiment B) and \u003cem\u003eRhizophagus\u003c/em\u003e (experiment E). Among the 27 differentially expressed NB genes, 13 belonged to the category containing the three motifs, which are probably functional in activating responses.\u003c/p\u003e\u003cp\u003eIn particular, genes such as 26 (Manes_09G025400), 34 (Manes_09G024872), and 163 (Manes_03G060801), were localized at the periphery of the biplot, indicating an important contribution of their differential expression to the plant response. Several recent reports have shown the induction of particular NB genes in response to pathogens (Zhang et al., 2015; Yu et al., 2021; Kan et al., 2024; Shi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTaken together, these results and previous ones reinforce the importance of NB in mounting a response and that they can be regulated at the transcriptional level. Previous transcriptomic studies during cassava interactions with pathogens such as the virus CBSV (Maruthi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and Xpm (Mu\u0026ntilde;oz Bodnar et al., 2014) have not detected differentially expressed NB genes. In this work, we conducted two different strategies to identify DEGs. In the first approach, the information of all annotated genes was included, and in the second one, the analysis was conducted only considering the NB genes. Generally, more differentially expressed NB genes were detected when the second approach was used. This highlights once again that the low expression level of NB genes can mask their identification as DEGs.\u003c/p\u003e\u003cp\u003eBy combining different analyses, it was possible to gain a deep understanding of the structure, expression, diversity, and concerted action of the different NB proteins in cassava. The integrative information provided new putative functions for NB lacking complete motifs and underscored the importance of transcriptional regulation of NB genes in responding to different stimuli, notably biotic responses.\u003c/p\u003e\u003cp\u003eFrom this analysis, it was possible to identify NB genes that can be considered strong candidates for further functional validation and potential employment in breeding programs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eOn behalf of all authors, the corresponding author states that there is no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCEL and LLK conceptualized the study, analyzed the data and wrote the manuscript. MAB, DSP and MAR collected, analyzed the data and prepared the figures.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThe authors would like to thank the students of the course in Statistical Genomic of the Statistics Department of Universidad Nacional de Colombia \u0026ndash; sede Bogot\u0026aacute; Juan Felipe Avella Duque and Diana Carolina Bri\u0026ntilde;ez Perales for their participation in the first transcriptomic data analysis used for this work and all members of the Manihot Biotec research group from Universidad Nacional de Colombia \u0026ndash; sede Bogot\u0026aacute; for their input during the development of the study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study are already available at NCBI (PRJNA257332, PRJNA688032, PRJNA491633, PRJNA396755, PRJNA911587 and GSE82279, GSE234712, GSE53369, GSE141125)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBoutrot F, Zipfel C (2017) Function, discovery, and exploitation of plant pattern recognition receptors for broad-spectrum disease resistance. Annu Rev Phytopathol 55:257\u0026ndash;286\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCock J, Connor D (2021) Cassava. In: Sadras VO, Calderini DF (eds) Crop Physiology Case Histories for Major Crops. Academic, Amsterdam, pp 588\u0026ndash;633\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eContreras MP, L\u0026uuml;dke D, Pai H, Toghani A, Kamoun S (2023a) NLR receptors in plant immunity: making sense of the alphabet soup. EMBO Rep 24:e57495\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeyoung BJ, Innes RW (2006) Plant NBS\u0026ndash;LRR proteins in pathogen sensing and host defense. Nat Immunol 7:1243\u0026ndash;1249\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHussain A, Khan AA, Aslam MQ et al (2024) Comparative analysis, diversification, and functional validation of plant nucleotide-binding site domain genes. 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Bioinformatics 23:2947\u0026ndash;2948\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu Z, Xie J, Wang H, Zhong X, Li H, Yu J, Kang J (2019) Identification and expression profiling analysis of NBS-LRR genes involved in Fusarium oxysporum f.sp. conglutinans resistance in cabbage. 3 Biotech 9:202\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLozano R, Hamblin MT, Prochnik S, Jannink JL (2015) Identification and distribution of the NBS-LRR gene family in the Cassava genome. BMC Genomics 16:360\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLukasik E, Takken FL (2009) STANDing strong, resistance proteins instigators of plant defence. Curr Opin Plant Biol 12:427\u0026ndash;436\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaruthi MN, Bouvaine S, Tufan HA, Mohammed IU, Hillocks RJ (2014) Transcriptional Response of Virus-Infected Cassava and Identification of Putative Sources of Resistance for Cassava Brown Streak Disease. 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Mol Plant Pathol 22:1520\u0026ndash;1537\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7624510/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7624510/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProteins containing the NB (Nucleotide Binding) domain are intracellular receptors or co-receptors that recognize proteins from microorganisms directly or indirectly. Plant genomes contain a variable number of NB genes, but the function of most of them remains unknown. Cassava is a crucial staple crop, and given its drought-tolerant nature, it is a promising crop for addressing climate change. Here, we describe the manual curation of 262 NB genes present in the cassava genome. The corresponding proteins were classified according to the presence of additional domains such as TIR (Toll Interleukin-1 Receptor) and/or LRR (Leucine Rich Repeat). The gene expression of these genes was evaluated using several transcriptomic experiments available in databases. Our analysis revealed that most NB genes are expressed at low or very low levels, with around 20% of them showing high expression values. The differential expression analysis detected 26 differentially expressed NB genes of which nine correspond to NB proteins lacking one or two motifs present in the NB domain. An integrative approach that took into account gene expression, polymorphisms level, and co-expression network metrics demonstrated that some NB proteins lacking certain motifs play important roles in the network structure, despite their low expression. On the other hand, the same analysis highlights the importance of complete NB proteins that showed higher levels of fold change. Several NB genes represent excellent candidates for further functional validation studies and/or breeding programs.\u003c/p\u003e","manuscriptTitle":"Genomics of the NB genes in cassava: from gene expression to integrative biology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 19:05:44","doi":"10.21203/rs.3.rs-7624510/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d8536302-ca79-47c3-9a91-37becd2bbdea","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-12T11:11:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-24 19:05:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7624510","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7624510","identity":"rs-7624510","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}