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.

Genomic analyses of heat stress transcription factors (HSFs) in simulated drought stress response and storage root deterioration after harvest in cassava.

Heat shock factors (HSFs) play crucial roles in various plant stress responses. However, the current knowledge about HSFs in cassava, an important crop, is still insufficient. In this research, we identified 32 cassava HSF genes (MeHSFs) and clustered them into three groups (A, B, C) based on phylogenetic analysis and structural characteristics. Conserved motif analyses showed that MeHSFs display domains characteristic to HSF transcription factors. Gene structure analyses suggested that 29 MeHSFs contained only two exons. All identified 32 cassava MeHSFs were distributed on 13 chromosomes. Their expression profiles revealed that the different MeHSFs were expressed differentially in different tissues, most high expression genes belonged to group A. The similar MeHSFs were up-regulated after treatment with both PEG and abscisic acid (ABA), which implied that these MeHSFs may participate in resistance to simulated drought stress associated with the ABA signaling pathway. In addition, several MeHSFs were induced during postharvest physiological deterioration (PPD) in cassava. Our results provided basic but important knowledge for future gene function analysis of MeHSFs toward efforts in improving tolerance to abiotic stress and PPD in cassava.

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

BACKGROUND: Aquaporins (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. RESULTS: This 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. CONCLUSIONS: Our 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.

Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz).

BACKGROUND: Cassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced. METHODS: Fully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization. RESULTS: In this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 107 protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H+/monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system. CONCLUSION: We optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.

Physiological Investigation and Transcriptome Analysis of Polyethylene Glycol (PEG)-Induced Dehydration Stress in Cassava.

Cassava is an important tropical and sub-tropical root crop that is adapted to drought environment. However, severe drought stress significantly influences biomass accumulation and starchy root production. The mechanism underlying drought-tolerance remains obscure in cassava. In this study, changes of physiological characters and gene transcriptome profiles were investigated under dehydration stress simulated by polyethylene glycol (PEG) treatments. Five traits, including peroxidase (POD) activity, proline content, malondialdehyde (MDA), soluble sugar and soluble protein, were all dramatically induced in response to PEG treatment. RNA-seq analysis revealed a gradient decrease of differentially expressed (DE) gene number in tissues from bottom to top of a plant, suggesting that cassava root has a quicker response and more induced/depressed DE genes than leaves in response to drought. Overall, dynamic changes of gene expression profiles in cassava root and leaves were uncovered: genes related to glycolysis, abscisic acid and ethylene biosynthesis, lipid metabolism, protein degradation, and second metabolism of flavonoids were significantly induced, while genes associated with cell cycle/organization, cell wall synthesis and degradation, DNA synthesis and chromatin structure, protein synthesis, light reaction of photosynthesis, gibberelin pathways and abiotic stress were greatly depressed. Finally, novel pathways in ABA-dependent and ABA-independent regulatory networks underlying PEG-induced dehydration response in cassava were detected, and the RNA-Seq results of a subset of fifteen genes were confirmed by real-time PCR. The findings will improve our understanding of the mechanism related to dehydration stress-tolerance in cassava and will provide useful candidate genes for breeding of cassava varieties better adapted to drought environment.

Molecular cloning and biochemical characterization of a novel cystatin from Hevea rubber latex.

A novel cDNA encoding a cysteine proteinase inhibitor or phytocystatin was isolated from Hevea brasiliensis RRIM600 rubber latex cDNA library. The full-length HbCPI obtained from rapid amplification of cDNA ends contains 588 bp. An open reading frame of 306 bp encodes for a protein of 101 amino acids with the typical inhibitory motifs of phytocystatin superfamily, namely the central signature motif QXVXG, a GG doublet and LARFAV-like motifs in the N-terminal part, and conserved A/PW residues in the C-terminal region. Sequence comparison showed that the deduced amino acid sequence was similar to that of cysteine protease inhibitor from Manihot esculenta (84% identity). The HbCPI was subcloned into expression vector pQE-40 and then overexpressed in Escherichia coli M15 strain (pREP4) as a His-tagged recombinant protein with molecular mass approximately 13 kDa. The purified HbCPI showed thermal stable property and efficiently inhibited the protease activity of papain by non-competitive inhibition with K(i) value of 15.4 nM. Beside latex, HbCPI also transcripted in leaf and young seed. The HbCPI message accumulation was induced by phytopathogenic fungi Phytophthora palmivora infection. These data suggest that HbCPI might play crucial roles in defense mechanism against biotic stimuli.