Cassava Genes Research Articles

TAL Effector Repertoires of Strains of Xanthomonas phaseoli pv. manihotis in Commercial Cassava Crops Reveal High Diversity at the Country Scale.

Transcription activator-like effectors (TALEs) play a significant role for pathogenesis in several xanthomonad pathosystems. Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of Cassava Bacterial Blight (CBB), uses TALEs to manipulate host metabolism. Information about Xpm TALEs and their target genes in cassava is scarce, but has been growing in the last few years. We aimed to characterize the TALE diversity in Colombian strains of Xpm and to screen for TALE-targeted gene candidates. We selected eighteen Xpm strains based on neutral genetic diversity at a country scale to depict the TALE diversity among isolates from cassava productive regions. RFLP analysis showed that Xpm strains carry TALomes with a bimodal size distribution, and affinity-based clustering of the sequenced TALEs condensed this variability mainly into five clusters. We report on the identification of 13 novel variants of TALEs in Xpm, as well as a functional variant with 22 repeats that activates the susceptibility gene MeSWEET10a, a previously reported target of TAL20Xam668. Transcriptomics and EBE prediction analyses resulted in the selection of several TALE-targeted candidate genes and two potential cases of functional convergence. This study provides new bases for assessing novel potential TALE targets in the Xpm–cassava interaction, which could be important factors that define the fate of the infection.

Molecular cloning and expression analysis of sucrose phosphate synthase genes in cassava (Manihot esculenta Crantz)

Sucrose phosphate synthase (SPS), a key rate-limiting enzyme in the sucrose biosynthesis pathway in plants, is encoded by a multi-gene family. Until recently, the identification and characterization of the SPS gene family have been performed for dozens of plant species; however, few studies have involved a comprehensive analysis of the SPS family members in tropical crops, such as cassava (Manihot esculenta Crantz). In the current study, five SPS genes (MeSPS1, MeSPS2, MeSPS3, MeSPS4, and MeSPS5) were isolated from cassava, and their sequence characteristics were comprehensively characterized. These MeSPS genes were found distributed on five chromosomes (Chr2, Chr14, Chr15, Chr16, and Chr18). Phylogenetic analysis showed that the MeSPS protein sequences were clustered into three families, together with other SPS sequences from both dicot and monocot species (families A, B, and C). The spatio-temporal expression pattern analysis of MeSPS genes showed a tissue-specific and partially overlapping expression pattern, with the genes mainly expressed in source tissues during cassava growth and development. Correlation analysis revealed that the expression of MeSPS genes correlated positively with root starch content, indicating that the expression of MeSPS genes might accelerate the rate of starch accumulation in the roots of cassava plants.

Enhancing of anthracnose disease resistance indicates a potential role of antimicrobial peptide genes in cassava

Cassava (Manihot esculenta Crantz.) is an important economic crop in tropical countries. Demands for using cassava in food, feed and biofuel industries have been increasing worldwide. Cassava anthracnose disease, caused by Colletotrichum gloeosporioides f.sp. manihotis (CAD), is considered a major problem in cassava production. To minimize the effects of such disease, this study investigated the response of cassava to attack by CAD and how the plants defend themselves against this threat. Genome-wide identification of antimicrobial peptide genes (AMPs) and their expression in response to fungal infection was performed in the resistant cassava cultivar (Huay Bong 60; HB60) in comparison with the highly susceptible cultivar (Hanatee; HN). A total of 114 gene members of AMP were identified in the cassava genome database. Fifty-six gene members were selected for phylogenetic tree construction and analysis of putative cis-acting elements in their promoter regions. Differential expression profiles of six candidate genes were observed in response to CAD infection of both cassava cultivars. Upregulation of snakins, MeSN1 and MeSN2 was found in HB60, whereas MeHEL, Me-AMP-D2 and MeLTP2 were highly induced in HN. The MeLTP1 gene was not expressed in either cultivar. HB60 showed a reduced severity rating in comparison to HN after CAD infection. The biomembrane permeability test of fungal CAD was strongly affected after treatment with protein extract derived from CAD-infected HB60. Altogether, these findings suggest that snakins have a potential function in the CAD defense response in cassava. These results could be useful for cassava improvement programs to fight fungal pathogen.

Analysis Of ZDS and LCYb Enzyme Coding Gene Related To Beta Carotene Bioshynthesis in Cassava (Manihot esculenta Crantz) using Reverse transcription PCR (RT-PCR)

This study aims to determine variations in the expression of ZDS and LCYb enzymes encoding several types of yellow tubers. Cassava used, namely Carvita 25, Nangka and Mentega 2 (yellow bulb) and Menti (white bulb) were used as controls. The target enzyme coding genes (ZDS and LCYb) in cassava were identified in an online database, Phytozome using the BLAST method. Design and primary analysis for the target genes was carried out using online software, OligoAnalyzer and PCR. The expression of the target enzyme coding gene was analyzed using the Reverse Transcription PCR (RT-PCR) method. The BLAST results in the cassava genome in the Phytozome database showed that the enzymes ZDS and LCYb in cassava were encoded by two genes. The primary optimization results showed the primary annealing temperature for the LCYb enzyme coding gene was 55 ° C. The ZDS primers were not amplified after several replications, so that only the LCYb enzyme coding gene was selected for qualitative gene expression analysis using RT-PCR. A positive control in the analysis of gene expression is the housekeeping gene, PP2A. Electrophoresis results of PCR products (RT-PCR) showed negative results (no DNA bands were detected) in all samples from both yellow and white tuber samples and its housekeeping gene. This is possible from the low quality of RNA used in cDNA synthesis.

Genome-wide analysis and phosphorylation sites identification of the 14-3-3 gene family and functional characterization of MeGRF3 in cassava.

The biological functionality of many members of the 14-3-3 gene family is regulated via phosphorylation at multiple amino acid residues. The specific phosphorylation-mediated regulation of these proteins during cassava root tuberization, however, is not well understood. In this study, 15 different 14-3-3 genes (designated MeGRF1 - 15) were identified within the cassava genome. Based upon evolutionary conservation and structural analyses, these cassava 14-3-3 proteins were grouped into ε and non-ε clusters. We found these 15 MeGRF genes to be unevenly distributed across the eight cassava chromosomes. When comparing the expression of these genes during different developmental stages, we found that three of these genes (MeGRF9, 12 and 15) were overexpressed at all developmental stages at 75, 104, 135, 182 and 267 days post-planting relative to the fibrous root stage, whereas two (MeGRF5 and 7) were downregulated during these same points. In addition, the expression of most MeGRF genes changed significantly in the early and middle stages of root tuberization. This suggests that these different MeGRF genes likely play distinct regulatory roles during cassava root tuberization. Subsequently, 18 phosphorylated amino acid residues were detected on nine of these MeGRF proteins. A phosphomimetic mutation at serine-65 in MeGRF3 in Arabidopsis thaliana (Arabidopsis) slightly influenced starch metabolism in these plants, and significantly affected the role of MeGRF3 in salt stress responses. Together these results indicate that 14-3-3 genes play key roles in responses to abiotic stress and the regulation of starch metabolism, offering valuable insights into the functions of these genes in cassava.

Variation in transcriptional profiles of carotenoid biosynthetic genes in Indonesian yellow- and white-fleshed tuberous root cassava (Manihot esculenta Crantz) accessions

Root-tubers of cassava (Manihot esculenta Crantz) accumulate high starchy carbohydrates, protein, and carotenoids. Carotenoids were associated with tuber colorus, i.e., yellow-fleshed tuber accumulates more carotenoids than white-fleshed root-tubers. Carotenoids serve nutrition for health, such as an antioxidant and provitamin A. This study aimed to analyze relative expressions (RE) of carotenoid genes in Indonesian yellow and white cassava (IYC and IWC, respectively). Nine-month-old fresh root-tubers of IYC (Adira-1, Mentega-2, and Ubi Kuning) and IWC (Adira-4 and Menti) were used for the analysis. RE of five genes: PSY (phytoene synthase), CRTISO (carotenoid isomerase), LYCa (α-lycopene cyclase), LYCb (β-lycopene cyclase), and BCH1 (beta carotene hydroxylase1), and a reference gene, i.e., EF1-A (elongation factor 1-alpha), were analyzed using a qRT-PCR. Results showed that RE of all genes were detected in IYC and IWC. In IWC, all genes were expressed high, suggesting biosynthetic fluxes through yellow α-carotenoids and xanthophylls. In YWC, variations on RE levels of all genes were observed. LYCa and LYCb were expressed high in Adira-1 and Mentega-2, indicating α- and β-carotenoids accumulate in root-tubers. However, BCH1 was expressed 2-3-folds lower in both accessions than all IWC, suggesting low zeaxanthin production and accumulation of β-carotene. In Ubi Kuning, α-carotenoids accumulation may not exist due to low LYCa RE. Also, higher BCH1 expression levels were observed in Ubi Kuning, suggesting a substantial zeaxanthin accumulation. Results are potential for improving carotenoid accumulation through breeding programs in cassava.

Genome-wide analyses of cassava Pathogenesis-related (PR) gene families reveal core transcriptome responses to whitefly infestation, salicylic acid and jasmonic acid

BackgroundWhiteflies are a threat to cassava (Manihot esculenta), an important staple food in many tropical/subtropical regions. Understanding the molecular mechanisms regulating cassava’s responses against this pest is crucial for developing control strategies. Pathogenesis-related (PR) protein families are an integral part of plant immunity. With the availability of whole genome sequences, the annotation and expression programs of the full complement of PR genes in an organism can now be achieved. An understanding of the responses of the entire complement of PR genes during biotic stress and to the defense hormones, salicylic acid (SA) and jasmonic acid (JA), is lacking. Here, we analyze the responses of cassava PR genes to whiteflies, SA, JA, and other biotic aggressors.ResultsThe cassava genome possesses 14 of the 17 plant PR families, with a total of 447 PR genes. A cassava PR gene nomenclature is proposed. Phylogenetic relatedness of cassava PR proteins to each other and to homologs in poplar, rice and Arabidopsis identified cassava-specific PR gene family expansions. The temporal programs of PR gene expression in response to the whitefly (Aleurotrachelus socialis) in four whitefly-susceptible cassava genotypes showed that 167 of the 447 PR genes were regulated after whitefly infestation. While the timing of PR gene expression varied, over 37% of whitefly-regulated PR genes were downregulated in all four genotypes. Notably, whitefly-responsive PR genes were largely coordinately regulated by SA and JA. The analysis of cassava PR gene expression in response to five other biotic stresses revealed a strong positive correlation between whitefly and Xanthomonas axonopodis and Cassava Brown Streak Virus responses and negative correlations between whitefly and Cassava Mosaic Virus responses. Finally, certain associations between PR genes in cassava expansions and response to biotic stresses were observed among PR families.ConclusionsThis study represents the first genome-wide characterization of PR genes in cassava. PR gene responses to six biotic stresses and to SA and JA are demonstrably different to other angiosperms. We propose that our approach could be applied in other species to fully understand PR gene regulation by pathogens, pests and the canonical defense hormones SA and JA.

Involvement of abscisic acid-responsive element-binding factors in cassava (Manihot esculenta) dehydration stress response

Cassava (Manihot esculenta) is a major staple food, animal feed and energy crop in the tropics and subtropics. It is one of the most drought-tolerant crops, however, the mechanisms of cassava drought tolerance remain unclear. Abscisic acid (ABA)-responsive element (ABRE)-binding factors (ABFs) are transcription factors that regulate expression of target genes involved in plant tolerance to drought, high salinity, and osmotic stress by binding ABRE cis-elements in the promoter regions of these genes. However, there is little information about ABF genes in cassava. A comprehensive analysis of Manihot esculenta ABFs (MeABFs) described the phylogeny, genome location, cis-acting elements, expression profiles, and regulatory relationship between these factors and Manihot esculenta betaine aldehyde dehydrogenase genes (MeBADHs). Here we conducted genome-wide searches and subsequent molecular cloning to identify seven MeABFs that are distributed unevenly across six chromosomes in cassava. These MeABFs can be clustered into three groups according to their phylogenetic relationships to their Arabidopsis (Arabidopsis thaliana) counterparts. Analysis of the 5′-upstream region of MeABFs revealed putative cis-acting elements related to hormone signaling, stress, light, and circadian clock. MeABF expression profiles displayed clear differences among leaf, stem, root, and tuberous root tissues under non-stress and drought, osmotic, or salt stress conditions. Drought stress in cassava leaves and roots, osmotic stress in tuberous roots, and salt stress in stems induced expression of the highest number of MeABFs showing significantly elevated expression. The glycine betaine (GB) content of cassava leaves also was elevated after drought, osmotic, or salt stress treatments. BADH1 is involved in GB synthesis. We show that MeBADH1 promoter sequences contained ABREs and that MeBADH1 expression correlated with MeABF expression profiles in cassava leaves after the three stress treatments. Taken together, these results suggest that in response to various dehydration stresses, MeABFs in cassava may activate transcriptional expression of MeBADH1 by binding the MeBADH1 promoter that in turn promotes GB biosynthesis and accumulation via an increase in MeBADH1 gene expression levels and MeBADH1 enzymatic activity. These responses protect cells against dehydration stresses by preserving an osmotic balance that enhances cassava tolerance to dehydration stresses.

Using in vitro plants to study the cassava response to Xanthomonas phaseoli pv. manihotis infection

Cassava Bacterial Blight (CBB), caused by Xanthomonas phaseoli pv. manihotis (Xpm), is the most important bacterial disease of cassava (Manihot esculenta). Xpm employs Transcription Activator-Like effectors (TALE) to induce the expression of host susceptibility (S) genes favoring bacterial growth. The identification of resistant and susceptible cassava varieties as well as the identification of the cassava gene MeSweet10a as a target of the effector TAL20, have been conducted on plants grown from mature stem-cuttings. We explored the possibility of using in vitro plants to study cassava-Xpm interactions. Cassava in vitro plants of varieties 60444 and CM6438–14 were susceptible and resistant, respectively, to infection by strain Xpm668. In addition, the expression of MeSweet10a was induced in the susceptible but not in the resistant variety, which was not associated with polymorphisms in the Effector Binding Element (EBE) reported for TAL20. Three other Xpm strains, Xpm531, Xpm681 and Xpm1061, were also able to induce the TAL20 cognate target gene in the susceptible cassava variety 60444. These results demonstrate the usefulness of in vitro plants to study the phenotypic and molecular responses of cassava during Xpm infection.

The Class III Peroxidase (POD) Gene Family in Cassava: Identification, Phylogeny, Duplication, and Expression.

The class III peroxidase (POD) enzymes participate in plant development, hormone signaling, and stress responses. However, little is known about the POD family in cassava. Here, we identified 91 cassava POD genes (MePODs) and classified them into six subgroups using phylogenetic analysis. Conserved motif analysis demonstrated that all MePOD proteins have typical peroxidase domains, and gene structure analysis showed that MePOD genes have between one and nine exons. Duplication pattern analysis suggests that tandem duplication has played a role in MePOD gene expansion. Comprehensive transcriptomic analysis revealed that MePOD genes in cassava are involved in the drought response and postharvest physiological deterioration. Several MePODs underwent transcriptional changes after various stresses and related signaling treatments were applied. In sum, we characterized the POD family in cassava and uncovered the transcriptional control of POD genes in response to various stresses and postharvest physiological deterioration conditions. These results can be used to identify potential target genes for improving the stress tolerance of cassava crops.

Arabidopsis LEC1 and LEC2 Orthologous Genes Are Key Regulators of Somatic Embryogenesis in Cassava

High genotype-dependent variation in friable embryogenic callus (FEC) induction and subsequent somaclonal variation constitute bottlenecks for the application and scaling of genetic transformation (GT) technology to more farmer- and industry-preferred cassava varieties. The understanding and identification of molecular factors underlying embryogenic development in cassava may help to overcome these constraints. Here, we described the Arabidopsis thaliana LEAFY COTYLEDON (LEC) LEC1 and LEC2 orthologous genes in cassava, designated as MeLEC1 and MeLEC2, respectively. Expression analyses showed that both, MeLEC1 and MeLEC2, are expressed at higher levels in somatic embryogenic (SE) tissues in contrast with differentiated mature tissues. The rapid expression increase of MeLEC genes at early SE induction times strongly suggests that they are involved in the transition from a somatic to an embryonic state, and probably, in the competence acquisition for SE development in cassava. The independent overexpression of the MeLEC genes resulted in different regenerated events with embryogenic characteristics such as MeLEC1OE plants with cotyledon-like leaves and MeLEC2OE plants with somatic-like embryos that emerged over the surface of mature leaves. Transcript increases of other embryo-specific regulating factors were also detected in MeLECOE plants, supporting their mutual interaction in the embryo development coordination. The single overexpression of MeLEC2 was enough to reprogram the vegetative cells and induce direct somatic embryogenesis, which converts this gene into a tool that could improve the recovery of transformed plants of recalcitrant genotypes. The identification of MeLEC genes contributes not only to improve our understanding of SE process in cassava, but also provides viable alternatives to optimize GT and advance in gene editing in this crop, through the development of genotype-independent protocols.

Genome-wide comparison reveals divergence of cassava and rubber aquaporin family genes after the recent whole-genome duplication

BackgroundAquaporins (AQPs) are a class of integral membrane proteins that facilitate the passive transport of water and other small solutes across biological membranes. Despite their importance, little information is available in cassava (Manihot esculenta), a perennial shrub of the Euphorbiaceae family that serves the sixth major staple crop in the world.ResultsThis study presents a genome-wide analysis of the AQP gene family in cassava. The family of 42 members in this species could be divided into five subfamilies based on phylogenetic analysis, i.e., 14 plasma membrane intrinsic proteins (PIPs), 13 tonoplast intrinsic proteins (TIPs), nine NOD26-like intrinsic proteins (NIPs), four X intrinsic proteins (XIPs), and two small basic intrinsic proteins (SIPs). Best-reciprocal-hit-based sequence comparison and synteny analysis revealed 34 orthologous groups (OGs) present in the Euphorbiaceae ancestor, and nearly one-to-one or two-to-one orthologous relationships were observed between cassava with rubber/physic nut, reflecting the occurrence of one so-called ρ recent whole-genome duplication (WGD) in the last common ancestor of cassava and rubber. In contrast to a predominant role of the ρ WGD on family expansion in rubber, cassava AQP duplicates were derived from the WGD as well as local duplication. Species-specific gene loss was also observed in cassava, which includes the entire NIP4 group and/or six OGs. Comparison of conserved motifs and gene expression profiles revealed divergence of paralogs in cassava as observed in rubber.ConclusionsOur findings will not only improve our knowledge on family evolution in Euphorbiaceae, but also provide valuable information for further functional analysis of AQP genes in cassava and rubber.

Dual RNA-seq reveals large-scale non-conserved genotype\u2009\xd7\u2009genotype-specific genetic reprograming and molecular crosstalk in the mycorrhizal symbiosis

Arbuscular mycorrhizal fungi (AMF) impact plant growth and are a major driver of plant diversity and productivity. We quantified the contribution of intra-specific genetic variability in cassava (Manihot esculenta) and Rhizophagus irregularis to gene reprogramming in symbioses using dual RNA-sequencing. A large number of cassava genes exhibited altered transcriptional responses to the fungus but transcription of most of these plant genes (72%) responded in a different direction or magnitude depending on the plant genotype. Two AMF isolates displayed large differences in their transcription, but the direction and magnitude of the transcriptional responses for a large number of these genes was also strongly influenced by the genotype of the plant host. This indicates that unlike the highly conserved plant genes necessary for the symbiosis establishment, most of the plant and fungal gene transcriptional responses are not conserved and are greatly influenced by plant and fungal genetic differences, even at the within-species level. The transcriptional variability detected allowed us to identify an extensive gene network showing the interplay in plant–fungal reprogramming in the symbiosis. Key genes illustrated that the two organisms jointly program their cytoskeleton organization during growth of the fungus inside roots. Our study reveals that plant and fungal genetic variation has a strong role in shaping the genetic reprograming in response to symbiosis, indicating considerable genotype × genotype interactions in the mycorrhizal symbiosis. Such variation needs to be considered in order to understand the molecular mechanisms between AMF and their plant hosts in natural communities.

Cloning and Expression Analysis of MeTCP4 Transcription Factor from Cassava and Construction of Plant Expression Vector

The TCP transcription factor family genes are involved in multiple regulation of plant growth and environmental stress. However, these family genes are rarely studied in cassava (Manihot esculenta). There are 36 TCP genes in cassava. Phylogenetic tree analysis indicates that MeTCP can be divided into 8 subgroups. MeTCP4 is one of 36 genes in cassava TCP family, which can be transcribed by miR319 and sheared. MeTCP4 contained an open reading frame (ORF) of 1 269 bp, which encoded 422 amino acids with a predicted molecular weight of 45.75 kD and theoretical PI of 6.17. The full-length cDNA sequence of MeTCP4 was cloned from the cassava genome. The results of quantitative RT-PCR showed that the expression of MeTCP4 can be found in all the tested tissues, with the lowest in roots, second in stems and the highest in leaves. MeTCP4 gene was repressed by drought and low temperature stress. Construction of high and low plant expression vector would provide basis for further study of the function of the gene.

Genome-wide identification and characterization of the Dof gene family in cassava (Manihot esculenta)

Dof proteins comprise a plant-specific transcription factor family involved in regulation of many plant processes. Despite their diverse roles, little information is available in cassava (Manihot esculenta, Euphorbiaceae), the sixth major staple crop in the world. This study presents the first genome-wide identification of Dof family genes in cassava, resulting in 45 family members that are unevenly distributed across 17 out of the 18 chromosomes. Phylogenetic analysis assigned these genes into eight groups representing four subfamilies. Synteny analysis and BRH (Best Reciprocal Hit) method were also adopted to study evolutionary relationships. Among 21 MeDof duplicates identified, the majority of them were derived from the recent whole-genome duplication (WGD), whereas only one from local duplication. Transcriptional profiling revealed diverse expression patterns among various tissues, and further comparison of conserved motifs and expression profiles revealed divergence of MeDof duplicates. These findings provide valuable information for functional analysis of Dof genes in cassava.

Genome-Wide Identification and Expression Profiling Analysis of the Galactinol Synthase Gene Family in Cassava (Manihot esculenta Crantz)

Galactinol synthases (GolSs) are the key enzymes that participate in raffinose family oligosaccharides (RFO) biosynthesis, which perform a big role in modulating plant growth and response to biotic or abiotic stresses. To date, no systematic study of this gene family has been conducted in cassava (Manihot esculenta Crantz). Here, eight MeGolS genes are isolated from the cassava genome. Based on phylogenetic background, the MeGolSs are clustered into four groups. Through predicting the cis-elements in their promoters, it was discovered that all MeGolS members act as hormone-, stress-, and tissue-specific related elements to different degrees. MeGolS genes exhibit incongruous expression patterns in various tissues, indicating that different MeGolS proteins might have diverse functions. MeGolS1 and MeGolS3–6 are highly expressed in leaves and midveins. MeGolS3–6 are highly expressed in fibrous roots. Quantitative real-time Polymerase Chain Reaction (qRT-PCR) analysis indicates that several MeGolSs, including MeGolS1, 2, 5, 6, and 7, are induced by abiotic stresses. microRNA prediction analysis indicates that several abiotic stress-related miRNAs target the MeGolS genes, such as mes-miR156, 159, and 169, which also respond to abiotic stresses. The current study is the first systematic research of GolS genes in cassava, and the results of this study provide a basis for further exploration the functional mechanism of GolS genes in cassava.

Phylogenetic Analysis and Expression Profiles of Starch Phosphorylase Family Genes in Cassava under Diverse Environments

Starch phosphorylase (Pho), especially the plastidial type, plays an important role in starch biosynthesis. The availability of a complete cassava (Manihot esculenta Crantz) genome database allowed the identification of five Pho family members, which could be grouped into the plastidial type (MePho1a, MePho1b, and MePho1c) and cytosolic type (MePho2 and MePho3) on the basis of phylogenetic analysis. Spatial gene expression analysis of various cassava tissues revealed that plastidial MePho1b had the highest transcriptional activity in root stele, whereas the cytosolic MePho2 was expressed with higher levels in leaves and petiole. Spatial expression analysis showed that MePho1b was strongly expressed in the tuber during root development process, especially at 70 and 180 d after planting, whereas MePho1a had highest expression in leaves at 180 d after planting. Expression of MePho1b and MePho2 could be induced by salt, drought, exogenous abscisic acid, methyl jasmonate, and salicylic acid signal in the leaves of abiotic stress‐treated plantlets. In addition, the expression profiles of MePho1b and starch branching enzyme gene member MeSBE2.2 exhibited significant correlation under diverse stress environments, indicating that they may have coordinated functions.

Functional analysis of MeCIPK23 and MeCBL1/9 in cassava defense response against Xanthomonas axonopodis pv. manihotis.

MeCIPK23 interacts with MeCBL1/9, and they confer improved defense response, providing potential genes for further genetic breeding in cassava. Cassava (Manihot esculenta) is an important food crop in tropical area, but its production is largely affected by cassava bacterial blight. However, the information of defense-related genes in cassava is very limited. Calcium ions play essential roles in plant development and stress signaling pathways. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) are crucial components of calcium signals. In this study, systematic expression profile of 25MeCIPKs in response to Xanthomonas axonopodis pv. manihotis (Xam) infection was examined, by which seven candidate MeCIPKs were chosen for functional investigation. Through transient expression in Nicotiana benthamiana leaves, we found that six MeCIPKs (MeCIPK5, MeCIPK8, MeCIPK12, MeCIPK22, MeCIPK23 and MeCIPK24) conferred improved defense response, via regulating the transcripts of several defense-related genes. Notably, we found that MeCIPK23 interacted with MeCBL1 and MeCBL9, and overexpression of these genes conferred improved defense response. On the contrary, virus-induced gene silencing of either MeCIPK23 or MeCBL1/9 or both genes resulted in disease sensitive in cassava. To our knowledge, this is the first study identifying MeCIPK23 as well as MeCBL1 and MeCBL9 that confer enhanced defense response against Xam.

MeBIK1, a novel cassava receptor-like cytoplasmic kinase, regulates PTI response of transgenic Arabidopsis.

Cassava bacterial blight is the most destructive disease in cassava, causing a significant reduction in its production. The innate immunity response, which has a broad spectrum and a persistent effect, is the basal defence of plants in response to pathogens. Isolation and identification of innate immune-related genes in cassava will contribute to understanding the disease resistance mechanism. In Arabidopsis, the receptor-like cytoplasmic kinase (RLCK) AtBIK1 is known to be an important signal mediator in pathogen-associated molecular pattern-triggered immunity (PTI) response, forming a signal complex from various receptors including the flagellin receptor FLS2, the chitin receptor CERK1 and the receptor for bacterial EF-Tu EFR (Zhang et al. 2010). In the present study, we selected a candidate receptor-like cytoplasmic kinase gene, MeBIK1, from the cassava genome. MeBIK1 encodes a 409 amino acid polypeptide comprising a typical serine/threonine protein kinase domain, and is located on the cell membrane. MeBIK1 gene expression was significantly increased upon stimulation with flagellin (flg22) and peaked at 1h. In vitro genetic complementation experiment showed that MeBIK1 complemented the reduced pathogen-associated molecular pattern-triggered immunity (PTI) response in Arabidopsis bik1 mutant. Arabidopsis MeBIK1 overexpression lines OX1 demonstrated a strong resistance to Xanthomonas axonopodis pv. manihotis HN01, whereas its sensitivity to Pseudomonas syringae pv. tomato DC3000 was enhanced. The peak level of reactive oxygen species (ROS) burst was reached in different Arabidopsis plants (bik1, OX1 and wild type) at 12min after induction with flg22. However, the OX1 showed significantly higher ROS levels than the control and mutant, whereas the lowest level of ROS burst was found in the bik1 mutant. These results indicate that cassava MeBIK1 has a similar function as Arabidopsis AtBIK1 and improves disease resistance in transgenic Arabidopsis by regulating the PTI response.

Isolation and Characterization of Ftsz Genes in Cassava.

The filamenting temperature-sensitive Z proteins (FtsZs) play an important role in plastid division. In this study, three FtsZ genes were isolated from the cassava genome, and named MeFtsZ1, MeFtsZ2-1, and MeFtsZ2-2, respectively. Based on phylogeny, the MeFtsZs were classified into two groups (FtsZ1 and FtsZ2). MeFtsZ1 with a putative signal peptide at N-terminal, has six exons, and is classed to FtsZ1 clade. MeFtsZ2-1 and MeFtsZ2-2 without a putative signal peptide, have seven exons, and are classed to FtsZ2 clade. Subcellular localization found that all the three MeFtsZs could locate in chloroplasts and form a ring in chloroplastids. Structure analysis found that all MeFtsZ proteins contain a conserved guanosine triphosphatase (GTPase) domain in favor of generate contractile force for cassava plastid division. The expression profiles of MeFtsZ genes by quantitative reverse transcription-PCR (qRT-PCR) analysis in photosynthetic and non-photosynthetic tissues found that all of the MeFtsZ genes had higher expression levels in photosynthetic tissues, especially in younger leaves, and lower expression levels in the non-photosynthetic tissues. During cassava storage root development, the expressions of MeFtsZ2-1 and MeFtsZ2-2 were comparatively higher than MeFtsZ1. The transformed Arabidopsis of MeFtsZ2-1 and MeFtsZ2-2 contained abnormally shape, fewer number, and larger volume chloroplasts. Phytohormones were involved in regulating the expressions of MeFtsZ genes. Therefore, we deduced that all of the MeFtsZs play an important role in chloroplast division, and that MeFtsZ2 (2-1, 2-2) might be involved in amyloplast division and regulated by phytohormones during cassava storage root development.