Genomic and Expression Analysis of Cassava (Manihot esculenta Crantz) Chalcone Synthase Genes in Defense against Tetranychus cinnabarinus Infestation.

Cassava is susceptible to mites, especially Tetranychus cinnabarinus. Secondary metabolism products such as flavonoids play an important role as antimicrobial metabolites protecting plants against biotic stressors including fungal, pathogen, bacterial, and pest defense. The chalcone synthase (CHS) is the initial step of the phenylpropanoid pathway for producing flavonoids and is the gatekeeper of the pathway. Until recently, the CHS genes family has not been systematically studied in cassava. Thirty-nine CHS genes were identified from the cassava genome database. Based on phylogenetic and sequence composition analysis, these CHSs were divided into 3 subfamilies. Within the same subfamily, the gene structure and motif compositions of these CHS genes were found to be quite conserved. Duplication events, particularly segmental duplication of the cassava CHS genes, were identified as one of the main driving force of its expansion. Various cis-elements contained in the promoter might regulate the gene expression patterns of MeCHS. Protein-protein interaction (PPI) network analysis showed that MeCHS1 and MeCHS10 protein are more closely related to other family members. The expression of MeCHS genes in young leaves was higher than that in other tissues, and their expression varies even within the same tissue. Coincidentally, these CHS genes of most LAP subclasses were highly expressed in young leaves. The verified MeCHS genes showed consistent with the real-time reverse transcription quantitative PCR (RT-qPCR) and proteomic expression in protected and affected leaves respectively, indicating that these MeCHS genes play crucial roles in the response to T. cinnabarinus. This study is the first to comprehensively expatiate the information on MeCHS family members. These data will further enhance our understanding both the molecular mechanisms and the effects of CHS genes. In addition, the results will help to further clarify the effects on T. cinnabarinus and provide a theoretical basis for the potential functions of the specific CHS gene in resistance to mites and other biotic stress.

Exploiting DNA methylation in cassava under water deficit for crop improvement.

DNA methylation plays a key role in the development and plant responses to biotic and abiotic stresses. This work aimed to evaluate the DNA methylation in contrasting cassava genotypes for water deficit tolerance. The varieties BRS Formosa (bitter) and BRS Dourada (sweet) were grown under greenhouse conditions for 50 days, and afterwards, irrigation was suspended. The stressed (water deficit) and non-stressed plants (negative control) consisted the treatments with five plants per variety. The DNA samples of each variety and treatment provided 12 MethylRAD-Seq libraries (two cassava varieties, two treatments, and three replicates). The sequenced data revealed methylated sites covering 18 to 21% of the Manihot esculenta Crantz genome, depending on the variety and the treatment. The CCGG methylated sites mapped mostly in intergenic regions, exons, and introns, while the CCNGG sites mapped mostly intergenic, upstream, introns, and exons regions. In both cases, methylated sites in UTRs were less detected. The differentially methylated sites analysis indicated distinct methylation profiles since only 12% of the sites (CCGG and CCNGG) were methylated in both varieties. Enriched gene ontology terms highlighted the immediate response of the bitter variety to stress, while the sweet variety appears to suffer more potential stress-damages. The predicted protein-protein interaction networks reinforced such profiles. Additionally, the genomes of the BRS varieties uncovered SNPs/INDELs events covering genes stood out by the interactomes. Our data can be useful in deciphering the roles of DNA methylation in cassava drought-tolerance responses and adaptation to abiotic stresses.

In silico prediction of candidate gene targets for the management of African cassava whitefly (Bemisia tabaci, SSA1-SG1), a key vector of viruses causing cassava brown streak disease.

Whiteflies (Bemisia tabaci sensu lato) have a wide host range and are globally important agricultural pests. In Sub-Saharan Africa, they vector viruses that cause two ongoing disease epidemics: cassava brown streak disease and cassava mosaic virus disease. These two diseases threaten food security for more than 800 million people in Sub-Saharan Africa. Efforts are ongoing to identify target genes for the development of novel management options against the whitefly populations that vector these devastating viral diseases affecting cassava production in Sub-Saharan Africa. This study aimed to identify genes that mediate osmoregulation and symbiosis functions within cassava whitefly gut and bacteriocytes and evaluate their potential as key gene targets for novel whitefly control strategies. The gene expression profiles of dissected guts, bacteriocytes and whole bodies were compared by RNAseq analysis to identify genes with significantly enriched expression in the gut and bacteriocytes. Phylogenetic analyses identified three candidate osmoregulation gene targets: two α-glucosidases, SUC 1 and SUC 2 with predicted function in sugar transformations that reduce osmotic pressure in the gut; and a water-specific aquaporin (AQP1) mediating water cycling from the distal to the proximal end of the gut. Expression of the genes in the gut was enriched 23.67-, 26.54- and 22.30-fold, respectively. Genome-wide metabolic reconstruction coupled with constraint-based modeling revealed four genes (argH, lysA, BCAT & dapB) within the bacteriocytes as potential targets for the management of cassava whiteflies. These genes were selected based on their role and essentiality within the different essential amino acid biosynthesis pathways. A demonstration of candidate osmoregulation and symbiosis gene targets in other species of the Bemisia tabaci species complex that are orthologs of the empirically validated osmoregulation genes highlights the latter as promising gene targets for the control of cassava whitefly pests by in planta RNA interference.

Estimation of Genetic Diversity and Number of Unique Genotypes of Cassava Germplasm from Burkina Faso Using Microsatellite Markers.

Genetic diversity is very important in crop improvement. This study was carried out to assess the genetic diversity and the number of unique multilocus genotypes (MLGs) in a cassava collection in Burkina Faso. To achieve this objective, 130 cassava accessions were genotyped using 32 simple sequence repeat (SSR) markers. The results revealed that among these markers, twelve (12) were highly informative, with polymorphic information content (PIC) values greater than 0.50; twelve (12) were moderately informative, with PIC values ranging between 0.25 and 0.50; and eight (8) were not very informative, with PIC values lower than 0.25. A moderate level of genetic diversity was found for the population, indicated by the average expected heterozygosity (0.45) and the observed heterozygosity (0.48). About 83.8% of unique multilocus genotypes were found in the cassava collection, indicating that SSR markers seem to be most appropriate for MLG identification. Population structure analysis based on hierarchical clustering identified two subpopulations and the Bayesian approach suggested five clusters. Additionally, discriminant analysis of principal components (DAPC) separated the cassava accessions into 13 subpopulations. A comparison of these results and those of a previous study using single nucleotide polymorphisms (SNP) suggests that each type of marker can be used to assess the genetic structure of cassava grown in Burkina Faso.

Coat protein of cassava common mosaic virus targets RAV1 and RAV2 transcription factors to subvert immunity in cassava.

Cassava common mosaic virus (CsCMV, genus Potexvirus) is a prevalent virus associated with cassava mosaic disease, so it is essential to elucidate the underlying molecular mechanisms of the coevolutionary arms race between viral pathogenesis and the cassava (Manihot esculenta Crantz) defense response. However, the molecular mechanism underlying CsCMV infection is largely unclear. Here, we revealed that coat protein (CP) acts as a major pathogenicity determinant of CsCMV via a mutant infectious clone. Moreover, we identified the target proteins of CP-related to abscisic acid insensitive3 (ABI3)/viviparous1 (VP1) (MeRAV1) and MeRAV2 transcription factors, which positively regulated disease resistance against CsCMV via transcriptional activation of melatonin biosynthetic genes (tryptophan decarboxylase 2 (MeTDC2), tryptamine 5-hydroxylase (MeT5H), N-aceylserotonin O-methyltransferase 1 (MeASMT1)) and MeCatalase6 (MeCAT6) and MeCAT7. Notably, the interaction between CP, MeRAV1, and MeRAV2 interfered with the protein phosphorylation of MeRAV1 and MeRAV2 individually at Ser45 and Ser44 by the protein kinase, thereby weakening the transcriptional activation activity of MeRAV1 and MeRAV2 on melatonin biosynthetic genes, MeCAT6 and MeCAT7 dependent on the protein phosphorylation of MeRAV1 and MeRAV2. Taken together, the identification of the CP-MeRAV1 and CP-MeRAV2 interaction module not only illustrates a molecular mechanism by which CsCMV orchestrates the host defense system to benefit its infection and development but also provides a gene network with potential value for the genetic improvement of cassava disease resistance.

Cassava molecular genetics and genomics for enhanced resistance to diseases and pests.

Cassava (Manihot esculenta) is one of the most important sources of dietary calories in the tropics, playing a central role in food and economic security for smallholder farmers. Cassava production is highly constrained by several pests and diseases, mostly cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). These diseases cause significant yield losses, affecting food security and the livelihoods of smallholder farmers. Developing resistant varieties is a good way of increasing cassava productivity. Although some levels of resistance have been developed for some of these diseases, there is observed breakdown in resistance for some diseases, such as CMD. A frequent re-evaluation of existing disease resistance traits is required to make sure they are still able to withstand the pressure associated with pest and pathogen evolution. Modern breeding approaches such as genomic-assisted selection in addition to biotechnology techniques like classical genetic engineering or genome editing can accelerate the development of pest- and disease-resistant cassava varieties. This article summarizes current developments and discusses the potential of using molecular genetics and genomics to produce cassava varieties resistant to diseases and pests.

Identification of MeC3HDZ1/MeCNA as a potential regulator of cassava storage root development.

The storage root (SR) of cassava is the main staple food in sub-Saharan Africa, where it feeds over 500 million people. However, little is known about the genetic and molecular regulation underlying its development. Unraveling such regulation would pave the way for biotechnology approaches aimed at enhancing cassava productivity. Anatomical studies indicate that SR development relies on the massive accumulation of xylem parenchyma, a cell-type derived from the vascular cambium. The C3HDZ family of transcription factors regulate cambial cells proliferation and xylem differentiation in Arabidopsis and other species. We thus aimed at identifying C3HDZ proteins in cassava and determining whether any of them shows preferential activity in the SR cambium and/or xylem. Using phylogeny and synteny studies, we identified eight C3HDZ proteins in cassava, namely MeCH3DZ1-8. We observed that MeC3HDZ1 is the MeC3HDZ gene displaying the highest expression in SR and that, within that organ, the gene also shows high expression in cambium and xylem. In-silico analyses revealed the existence of a number of potential C3HDZ targets displaying significant preferential expression in the SR. Subsequent Y1H analyses proved that MeC3HDZ1 can bind canonical C3HDZ binding sites, present in the promoters of these targets. Transactivation assays demonstrated that MeC3HDZ1 can regulate the expression of genes downstream of promoters harboring such binding sites, thereby demonstrating that MeC3HDZ1 has C3HDZ transcription factor activity. We conclude that MeC3HDZ1 may be a key factor for the regulation of storage root development in cassava, holding thus great promise for future biotechnology applications.

Haplotype-resolved genome of heterozygous African cassava cultivar TMEB117 (Manihot esculenta).

Cassava (Manihot esculenta Crantz) is a vital tropical root crop providing essential dietary energy to over 800 million people in tropical and subtropical regions. As a climate-resilient crop, its significance grows as the human population expands. However, yield improvement faces challenges from biotic and abiotic stress and limited breeding. Advanced sequencing and assembly techniques enabled the generation of a highly accurate, nearly complete, haplotype-resolved genome of the African cassava cultivar TMEB117. It is the most accurate cassava genome sequence to date with a base-level accuracy of QV > 64, N50 > 35 Mbp, and 98.9% BUSCO completeness. Over 60% of the genome comprises repetitive elements. We predicted over 45,000 gene models for both haplotypes. This achievement offers valuable insights into the heterozygosity genome organization of the cassava genome, with improved accuracy, completeness, and phased genomes. Due to its high susceptibility to African Cassava Mosaic Virus (ACMV) infections compared to other cassava varieties, TMEB117 provides an ideal reference for studying virus resistance mechanisms, including epigenetic variations and smallRNA expressions.

Metabolic GWAS-based dissection of genetic basis underlying nutrient quality variation and domestication of cassava storage root.

BACKGROUND: Metabolites play critical roles in regulating nutritional qualities of plants, thereby influencing their consumption and human health. However, the genetic basis underlying the metabolite-based nutrient quality and domestication of root and tuber crops remain largely unknown. RESULTS: We report a comprehensive study combining metabolic and phenotypic genome-wide association studies to dissect the genetic basis of metabolites in the storage root (SR) of cassava. We quantify 2,980 metabolic features in 299 cultivated cassava accessions. We detect 18,218 significant marker-metabolite associations via metabolic genome-wide association mapping and identify 12 candidate genes responsible for the levels of metabolites that are of potential nutritional importance. Me3GT, MeMYB4, and UGT85K4/UGT85K5, which are involved in flavone, anthocyanin, and cyanogenic glucoside metabolism, respectively, are functionally validated through in vitro enzyme assays and in vivo gene silencing analyses. We identify a cluster of cyanogenic glucoside biosynthesis genes, among which CYP79D1, CYP71E7b, and UGT85K5 are highly co-expressed and their allelic combination contributes to low linamarin content. We find MeMYB4 is responsible for variations in cyanidin 3-O-glucoside and delphinidin 3-O-rutinoside contents, thus controlling SR endothelium color. We find human selection affects quercetin 3-O-glucoside content and SR weight per plant. The candidate gene MeFLS1 is subject to selection during cassava domestication, leading to decreased quercetin 3-O-glucoside content and thus increased SR weight per plant. CONCLUSIONS: These findings reveal the genetic basis of cassava SR metabolome variation, establish a linkage between metabolites and agronomic traits, and offer useful resources for genetically improving the nutrition of cassava and other root crops.

Integrative transcriptomics reveals association of abscisic acid and lignin pathways with cassava whitefly resistance.

BACKGROUND: Whiteflies are a global threat to crop yields, including the African subsistence crop cassava (Manihot esculenta). Outbreaks of superabundant whitefly populations throughout Eastern and Central Africa in recent years have dramatically increased the pressures of whitefly feeding and virus transmission on cassava. Whitefly-transmitted viral diseases threaten the food security of hundreds of millions of African farmers, highlighting the need for developing and deploying whitefly-resistant cassava. However, plant resistance to whiteflies remains largely poorly characterized at the genetic and molecular levels. Knowledge of cassava-defense programs also remains incomplete, limiting characterization of whitefly-resistance mechanisms. To better understand the genetic basis of whitefly resistance in cassava, we define the defense hormone- and Aleurotrachelus socialis (whitefly)-responsive transcriptome of whitefly-susceptible (COL2246) and whitefly-resistant (ECU72) cassava using RNA-seq. For broader comparison, hormone-responsive transcriptomes of Arabidopsis thaliana were also generated. RESULTS: Whitefly infestation, salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA) transcriptome responses of ECU72 and COL2246 were defined and analyzed. Strikingly, SA responses were largely reciprocal between the two cassava genotypes and we suggest candidate regulators. While susceptibility was associated with SA in COL2246, resistance to whitefly in ECU72 was associated with ABA, with SA-ABA antagonism observed. This was evidenced by expression of genes within the SA and ABA pathways and hormone levels during A. socialis infestation. Gene-enrichment analyses of whitefly- and hormone-responsive genes suggest the importance of fast-acting cell wall defenses (e.g., elicitor recognition, lignin biosynthesis) during early infestation stages in whitefly-resistant ECU72. A surge of ineffective immune and SA responses characterized the whitefly-susceptible COL2246's response to late-stage nymphs. Lastly, in comparison with the model plant Arabidopsis, cassava's hormone-responsive genes showed striking divergence in expression. CONCLUSIONS: This study provides the first characterization of cassava's global transcriptome responses to whitefly infestation and defense hormone treatment. Our analyses of ECU72 and COL2246 uncovered possible whitefly resistance/susceptibility mechanisms in cassava. Comparative analysis of cassava and Arabidopsis demonstrated that defense programs in Arabidopsis may not always mirror those in crop species. More broadly, our hormone-responsive transcriptomes will also provide a baseline for the cassava community to better understand global responses to other yield-limiting pests/pathogens.

Reduced glutathione and raffinose lengthens postharvest storage of cassava root tubers by improving antioxidant capacity and antibiosis.

Cassava is an ideal food security crop in marginal and drought environment. However, the post-harvest storage of cassava is urgent problem to be resolved. In this study, the storage tolerant and non-tolerant cassava were screened by measuring the change of Peroxidase (POD), Superoxide dismutase (SOD), Catalase (CAT) and Malondialdehyde (MDA) in seven cultivars of cassava. Compared with other cultivars, the cultivar of SC14 showed the highest level of SOD, MDA and POD respectively at 0 day, 12 day and 9 day postharvest while exhibited lowest level of CAT at 0 day postharvest, indicating the strongest antioxidant capability and storage tolerance. In contrast, GR15231, termed as storage non-tolerance cultivars, showed lowest SOD and POD at 12 day and kept a relative high level of CAT at 12 day post-harvest. In addition, SC14 has higher level of starch and dry substance than GR15231. Mass spectrum was performed for SC14 and GR15231 to explore the key metabolites regulating the storage tolerance of cassava. The results showed that the expression of glutathione (reduced) and raffinose was significantly decreased at 12 day post-harvest both in tolerant SC14 and non-tolerant GR15231. Compared with GR15231, SC14 showed higher level of raffinose both at 0 and 12 day post-harvest, indicating that raffinose may be the potential metabolites protecting SC14 cultivar from deterioration post-harvest. Additionally, raffinose ratio of SC14a/SC14b was five times less than that of GR15231a/GR15231b, reflecting the slower degradation of raffinose in SC14 cultivar compared with GR15231 cultivar. In conclusion, the antioxidant microenvironment induced by reduced glutathione and higher level of raffinose in SC14 cultivar might be the promising metabolites to improve its antioxidant capacity and antibiosis and thus maintained the quality of Cassava root tubers.

Genome-wide identification and expression analysis of heat shock protein gene family in cassava.

Heat shock proteins are important molecular chaperones that are involved in plant growth and stress responses. However, members of the Hsp family have been poorly studied in cassava. In this study, 225 MeHsp genes were identified in the cassava genome, and their genetic structures exhibited relatively conserved features within each subfamily. The 225 MeHsp genes showed random chromosomal distribution, and at least 74 pairs of segmentally duplicated MeHsp genes. Eleven tandemly duplicated MeHsp genes were identified. Cis-element analysis revealed the importance of MeHsps in plant adaptations to the environment. The prediction of protein interactions suggested that MeHsp70-20 may play a critical regulatory role in the interactive network. Furthermore, the expression profiles of MeHsps in different tissues and cell subsets were analyzed using bulk transcriptomics and single-cell transcriptomic data. Several subfamily genes exhibited unique expression patterns in the transcriptome and were selected for detailed analysis of the single-cell transcriptome. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed the expression patterns of these genes under temperature stress, further supporting the prediction of cis-acting elements. This study provides valuable information for understanding the functional characteristics of MeHsp genes and the evolutionary relationships between MeHsps.

Cluster-based photography and modeling integrated method for an efficient measurement of cassava leaf area.

Leaf area (LA) and biomass are important agronomic indicators of the growth and health of plants. Conventional methods for measuring the LA can be challenging, time-consuming, costly, and laborious, especially for a large-scale study. A hybrid approach of cluster-based photography and modeling was, thus, developed herein to improve practicality. To this end, data on cassava palmate leaves were collected under various conditions to cover a spectrum of viable leaf shapes and sizes. A total of 1,899 leaves from 3 cassava genotypes and 5 cultivation conditions were first assigned into clusters by size, based on their length (L) and width (W). Next, 111 representative leaves from all clusters were photographed, and data from image-processing were subsequently used for model development. The model based on the product of L and W outperformed the rest (R2 = 0.9566, RMSE = 20.00). The hybrid model was successfully used to estimate the LA of greenhouse-grown cassava as validation. This represents a breakthrough in the search for efficient, practical phenotyping tools for LA estimation, especially for large-scale experiments or remote fields with limited machinery.

Flowering induction in cassava using photoperiod extension premature pruning and plant growth regulators.

Cassava (Manihot esculenta Crantz) is a vital crop for food and economic security in many regions of the world. Despite the economic and social importance of cassava, challenges persist in developing superior varieties that meet the needs of farmers in terms of agronomic performance, nutritional quality, and resistance to pests and diseases. One of the main obstacles for genetic improvement is the lack of synchronization in flowering and the abortion of young flowers, making planned crosses and progeny production difficult. Therefore, the aim of this study was to evaluate the effect of photoperiod, premature pruning, and growth regulators on cassava flowering under low-altitude conditions in Brazil. Eight cassava clones with contrasting flowering capacity were assessed in Cruz das Almas, Bahia, using two photoperiods (ambient condition and extended photoperiod with red light for 12 hours), premature pruning at the first and second branching levels (with and without pruning), and the application of growth regulators: 0.5 mM 6-benzyladenine (BA) and 4.0 mM silver thiosulfate (STS) (with and without). Plots were assessed weekly for the number of female (NFF) and male (NMF) flowers, height of the first branching (H1B, in cm), number of days to the first branching (ND1B), and the number of branching events up to 240 days after planting (NOB). The extended photoperiod did not promote an increase in the number of flowers but allowed for precocity in cassava flowering, reducing the onset of flowering by up to 35 days, and significantly increasing the number of branches, which is closely related to flowering. The use of pruning and plant growth regulators (PGR) resulted in an increase in NFF from 2.2 (control) to 4.6 and NMF from 8.1 to 21.1 flowers. Therefore, under hot and humid tropical conditions at low altitudes in the Recôncavo of Bahia, manipulating the photoperiod and using premature pruning and plant growth regulators can accelerate cassava flowering, benefiting genetic improvement programs.

Correlate the cyanogenic potential and dry matter content of cassava roots and leaves grown in different environments.

Cassava (Manihot esculenta Crantz) is an essential stable food crop in Sub-Saharan Africa commonly consumed amongst the low-income communities in Africa. Though cassava roots and leaf have vast economic and commercial benefits, it produces cyanogenic glycosides, which are toxic and most often responsible for the bitter taste of some cassava cultivars. The study evaluates the cassava roots and leaves' cyanogenic potential and dry matter content of the Genetic Gain Assessment trial grown in a different environment. It establishes the association between the cyanogenic potential (CNP) and the roots and leaves dry matter (DM). Genetic Gain Assessment (GGA) cassava genotypes (N = 400) selected for the Uniform Yield Trial (UYT) breeding stage were planted under IVS (Dry season in Inland Valley Hydromorphic area) and Upland (rain-fed conditions) in two locations of IITA Research Farms, namely; Ibadan (IVS and Upland) and Mokwa (Upland) in Nigeria. The CNP content of cassava leaves in IVS, Mokwa, and Upland ranged from 3.39 to 272.16 mg/100 g, 4.28 to 228.72 mg/100 g, and 13.13 to 127.39 mg/100 g, respectively. However, the respective CNP range in root samples across IVS, Mokwa, and Upland was 0.76-76.31 mg/100 g, 0.94-136.53 mg/100 g, and 2.37-47.11 mg/100 g. Also, the mean ± SD of DM content of leaves were 27.97 ± 3.01%, 28.81 ± 4.01%, and 13.65 ± 3.69%, respectively, in IVS, Mokwa, and Upland, while the root samples had mean ± SD of DM content of 38.09 ± 4.80%, 32.69 ± ,5.93% and 24.63 ± 5.07% respectively. Furthermore, location and genotype had a highly significant effect (p < 0.001) on the CNP and DM of roots and leaves. Also, linear regressions were established between CNP and DM of root and leaf with regression equation; DM-Root = 1.1999*DM-Leaf (r = 0.956) and CNP-Root = 0.29006*CNP-Leaf (r = 0.54). The relationship between the DM (root and leaf) and CNP (root and leaf) could serve as a valuable "inter-prediction" tool for these parameters.

Single-cell RNA-sequencing profiles reveal the developmental landscape of the Manihot esculenta Crantz leaves.

Cassava (Manihot esculenta Crantz) is an important crop with a high photosynthetic rate and high yield. It is classified as a C3-C4 plant based on its photosynthetic and structural characteristics. To investigate the structural and photosynthetic characteristics of cassava leaves at the cellular level, we created a single-cell transcriptome atlas of cassava leaves. A total of 11,177 high-quality leaf cells were divided into 15 cell clusters. Based on leaf cell marker genes, we identified 3 major tissues of cassava leaves, which were mesophyll, epidermis, and vascular tissue, and analyzed their distinctive properties and metabolic activity. To supplement the genes for identifying the types of leaf cells, we screened 120 candidate marker genes. We constructed a leaf cell development trajectory map and discovered 6 genes related to cell differentiation fate. The structural and photosynthetic properties of cassava leaves analyzed at the single cellular level provide a theoretical foundation for further enhancing cassava yield and nutrition.

Genome-Wide Survey of the RWP-RK Gene Family in Cassava (Manihot esculenta Crantz) and Functional Analysis.

The plant-specific RWP-RK transcription factor family plays a central role in the regulation of nitrogen response and gametophyte development. However, little information is available regarding the evolutionary relationships and characteristics of the RWP-RK family genes in cassava, an important tropical crop. Herein, 13 RWP-RK proteins identified in cassava were unevenly distributed across 9 of the 18 chromosomes (Chr), and these proteins were divided into two clusters based on their phylogenetic distance. The NLP subfamily contained seven cassava proteins including GAF, RWP-RK, and PB1 domains; the RKD subfamily contained six cassava proteins including the RWP-RK domain. Genes of the NLP subfamily had a longer sequence and more introns than the RKD subfamily. A large number of hormone- and stress-related cis-acting elements were found in the analysis of RWP-RK promoters. Real-time quantitative PCR revealed that all MeNLP1-7 and MeRKD1/3/5 genes responded to different abiotic stressors (water deficit, cold temperature, mannitol, polyethylene glycol, NaCl, and H2O2), hormonal treatments (abscisic acid and methyl jasmonate), and nitrogen starvation. MeNLP3/4/5/6/7 and MeRKD3/5, which can quickly and efficiently respond to different stresses, were found to be important candidate genes for further functional assays in cassava. The MeRKD5 and MeNLP6 proteins were localized to the cell nucleus in tobacco leaf. Five and one candidate proteins interacting with MeRKD5 and MeNLP6, respectively, were screened from the cassava nitrogen starvation library, including agamous-like mads-box protein AGL14, metallothionein 2, Zine finger FYVE domain containing protein, glyceraldehyde-3-phosphate dehydrogenase, E3 Ubiquitin-protein ligase HUWE1, and PPR repeat family protein. These results provided a solid basis to understand abiotic stress responses and signal transduction mediated by RWP-RK genes in cassava.

Comparative analysis of salicylic acid levels and gene expression in resistant, tolerant, and susceptible cassava varieties following whitefly-mediated SLCMV infection.

Sri Lankan cassava mosaic virus (SLCMV), the primary pathogen responsible for cassava mosaic disease in cassava plantations, is transmitted via infected cutting stems and the whitefly vector, Bemisia tabaci. To obtain better insights into the defense mechanism of cassava against SLCMV, whiteflies were used to induce SLCMV infection for activating the salicylic acid (SA) signaling pathway, which triggers the innate immune system. The study aimed to investigate the specific interactions between viruliferous whiteflies and SA accumulation in resistant (C33), tolerant (Kasetsart 50; KU50), and susceptible (Rayong 11) cassava cultivars by infecting with SLCMV. Leaf samples were collected at various time points, from 1 to 7 days after inoculation (dai). The SA levels were quantified by gas chromatography-mass spectrometry and validated by quantitative reverse transcription polymerase chain reaction. The SA levels increased in KU50 and C33 plants at 2 and 3 dai, respectively, but remained undetected in Rayong11 plants. The expression of PR-9e, PR-7f5, SPS1, SYP121, Hsf8, and HSP90 increased in infected C33 plants at 4 dai, whereas that of KU50 plants decreased immediately at 2 dai, and that of Rayong11 plants increased at 1 dai but gradually decreased thereafter. These findings strongly indicate that SA plays a crucial role in regulating antiviral defense mechanisms, especially in SLCMV-resistant plants. Altogether, the findings provide valuable insights into the mechanisms underlying the activation of SA-mediated anti-SLCMV defense pathways, and the resistance, tolerance, and susceptibility of cassava, which can aid future breeding programs aimed at enhancing SLCMV resistance.

LESION SIMULATING DISEASE 3 regulates disease resistance via fine-tuning histone acetylation in cassava.

Bacterial blight seriously affects the growth and production of cassava (Manihot esculenta Crantz), but disease resistance genes and the underlying molecular mechanism remain unknown. In this study, we found that LESION SIMULATING DISEASE 3 (MeLSD3) is essential for disease resistance in cassava. MeLSD3 physically interacts with SIRTUIN 1 (MeSRT1), inhibiting MeSRT1-mediated deacetylation modification at the acetylation of histone 3 at K9 (H3K9Ac). This leads to increased H3K9Ac levels and transcriptional activation of SUPPRESSOR OF BIR1 (SOBIR1) and FLAGELLIN-SENSITIVE2 (FLS2) in pattern-triggered immunity, resulting in immune responses in cassava. When MeLSD3 was silenced, the release of MeSRT1 directly decreased H3K9Ac levels and inhibited the transcription of SOBIR1 and FLS2, leading to decreased disease resistance. Notably, DELLA protein GIBBERELLIC ACID INSENSITIVE 1 (MeGAI1) also interacted with MeLSD3, which enhanced the interaction between MeLSD3 and MeSRT1 and further strengthened the inhibition of MeSRT1-mediated deacetylation modification at H3K9Ac of defense genes. In summary, this study illustrates the mechanism by which MeLSD3 interacts with MeSRT1 and MeGAI1, thereby mediating the level of H3K9Ac and the transcription of defense genes and immune responses in cassava.