Cassava Tissues Research Articles

Initiation of Cassava Callus Culture and Its Prospect for Starch Production: A Systematic Mapping Study

Mass propagation of cassava on several hectares of arable land due to increasing demand for its starch is not feasible due to land availability, pests and disease invasion, and long cultivation period. Plant cell culture technology is a promising solution despite the scarcity of cassava callus culture for starch production applications. Therefore, a systematic mapping study (SMS) was performed to identify the applications of cassava tissue culture and its prospects in starch production and investigate the important parameters for cassava callus culture initiation. The SMS began with formulating research questions (RQs), conducting searches on various databases, collecting and screening related articles, and extracting and mapping the selected articles. A total of 56 of 589 articles in the initial searching phase were chosen to be used as references to answer each RQ. The extracted data indicates that cassava tissue culture was mostly used for micropropagation, while starch production from its tissue culture is still limited. Basal medium and plant growth regulators influence cassava callus culture initiation most. The findings of the SMS offer a better understanding of cassava tissue culture and the prospects of producing cassava starch.

Symplasmic phloem loading and subcellular transport in storage roots are key factors for carbon allocation in cassava.

Cassava (Manihot esculenta) is a deciduous woody perennial shrub that stores large amounts of carbon and water in its storage roots. Previous studies have shown that assimilating unloading into storage roots happens symplasmically once secondary anatomy is established. However, mechanisms controlling phloem loading and overall carbon partitioning to different cassava tissues remain unclear. Here, we used a combination of histological, transcriptional, and biochemical analyses on different cassava tissues and at different timepoints to better understand source-sink carbon allocation. We found that cassava likely utilizes a predominantly passive symplasmic phloem loading strategy, indicated by the lack of expression of genes coding for key players of sucrose transport, the existence of branched plasmodesmata in the companion cell/bundle sheath interface of minor leaf veins, and very high leaf sucrose concentrations. Furthermore, we showed that tissue-specific changes in anatomy and non-structural carbohydrate contents are associated with tissue-specific modification in gene expression for sucrose cleavage/synthesis, as well as subcellular compartmentalization of sugars. Overall, our data suggest that carbon allocation during storage root filling is mostly facilitated symplasmically and is likely mostly regulated by local tissue demand and subcellular compartmentalization.

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.

A system for rapid gene introgression into cassava immature leaves and subsequent recovery of transformed lines

Friable embryogenic callus (FEC) currently forms the tissue of choice for gene transfer to cassava tissues. However, this technology has not been sufficiently adopted in most laboratories, partly because of the challenging protocols involved in FEC production. Therefore, the development of transformation systems targeting readily accessible explants could enable the rapid improvement of this essential root tuber. This study developed a simple, efficient, and rapid transformation protocol for three elite cassava cultivars (TMS 60444, 08/354, KME 1) using immature leaf lobes (ILLs). Factors affecting callus and somatic embryo formation, such as auxin concentration and culture photoperiod, were optimized. Cassava transformation was achieved by co-cultivation of ILLs and callus with Agrobacterium strain EHA101 harboring pTF102 plasmid. We report a positive interactive effect between dark treatment and an increasing auxin concentration. Exposure to light was found to negatively affect the formation and viability of SEs by promoting tissue browning. The results further show that cassava transformation success depends on the cultivar, explant type, and age. Using this simple protocol, transgenic plants of TMS 60444, 08/354, and KME 1 with an average transformation frequency of 0.4% (0.5, 0.4, and 0.4%, respectively) were obtained in 6 months. Reverse transcription PCR, Southern blot, and histochemical GUS staining confirmed the stable integration of the gusA gene into the regenerated plantlets. This optimized transformation system for cassava is rapid, efficient, and simple to operate and will facilitate more genetic applications in cassava research.

Using bioenergy crop cassava (Manihot esculenta) for reclamation of heavily metal-contaminated land

Heavy metal contamination of agricultural lands may give rise to health risks by cultivation and consumption of food crops from such lands, as well as result in economic loss. Phytoremediation is an eco-friendly and cost-effective approach to restore contaminated soil. However, the restoration process is slow and its sustainability is difficult to maintain. Bioenergy crops may provide alternative economic benefits to agriculture sector and reduce the risks associated with transfering heavy metals into food webs. In this study, a field experiment was carried out to determine the level of reclamation that would be attained in severely heavy metal-contaminated land by planting cassava (Manihot esculenta), a bioenergy crop. The results showed that cassava could grow well on the derelict land, with a fresh tuber yield of 23.13–26.22 t ha−1 in one growing season, which could potentially produce 3680–4160 L ha−1 bioethanol. The economic income of the cassava was estimated to be 11.6–13.1 × 103 CNY ha−1. Among the cassava tissues, metal concentrations were lowest in the tuber. The soil fertility and acidity were ameliorated after cassava plantation, and the mobile and bioavailable metal fractions in the soils were decreased. The cultivation of cassava as a renewable energy crop appears applicable for sustainable utilization and reclamation of heavy metal-contaminated land.

Plant regeneration via protoplast electrofusion in cassava

Protoplast electrofusion between callus protoplasts of cultivar TMS60444 and mesophyll protoplasts of cultivar SC8 was performed as an approach for the genetic improvement of cassava. The fusion products were subsequently cultured in protoplast culture medium (TM2G) with gradual dilution for approximately 1–2 months. Then the protoplast-derived compact calli were transferred to suspension culture medium (SH) for suspension culture. The cultured products developed successively into embryos, mature embryos, and shoots on somatic embryo emerging medium (MSN), embryo maturation medium (CMM), and shoot elongation medium (CEM), respectively. And the shoots were then rooted on Murashige and Skoog (1962) medium (MS). Sixty-six cell lines were obtained and 12 of them developed into plantlets. Based on assessment of ploidy level and chromosome counting, four of these plantlets were tetraploid and the remaining eight were diploid. Based on assessment of ploidy level and simple sequence repeat (SSR) analysis, nine tetraploid cell lines, one diploid variant plant line and nine variant cell lines were obtained. The diploid variant plant line and the nine variant cell lines all showed partial loss of genetic material compared to that of the parent TMS60444, based on SSR patterns. These results showed that some new germplasm of cassava were created. In this study, a protocol for protoplast electrofusion was developed and validated. Another important conclusion from this work is the confirmation of a viable protocol for the regeneration of plants from cassava protoplasts. Going forward, we hope to provide technical guidance for cassava tissue culture, and also provide some useful inspiration and reference for further genetic improvement of cassava.

Cassava Metabolomics and Starch Quality.

Cassava plays an important role as a staple food for more than 800 million people in the world due to its ability to maintain relatively high productivity even in nutrient-depleted soils. Even though cassava has been the focus of several breeding programs and has become a strong focus of research in the last few years, relatively little is currently known about its metabolism and metabolic composition in different tissues. In this article, the absolute content of sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, carotenoids, chlorophylls, tocopherols, and total protein as well as starch quality is described based on multiple analytical techniques, with protocols specifically adjusted for material from different cassava tissues. Moreover, quantification of secondary metabolites relative to internal standards is presented using both non-targeted and targeted metabolomics approaches. The protocols have also been adjusted to apply to freeze-dried material in order to allow processing of field harvest samples that typically will require long-distance transport. © 2019 The Authors. Basic Protocol 1: Metabolic profiling by gas chromatography-mass spectrometry (GC-MS) Support Protocol 1: Preparation of freeze-dried cassava material Support Protocol 2: Preparation of standard compound mixtures for absolute quantification of metabolites by GC-MS Support Protocol 3: Preparation of retention-time standard mixture Basic Protocol 2: Determination of organic acids and phosphorylated intermediates by ion chromatography-mass spectrometry (IC-MS) Support Protocol 4: Preparation of standards and recovery experimental procedure Basic Protocol 3: Determination of soluble sugars, starch, and free amino acids Alternate Protocol: Determination of soluble sugars and starch Basic Protocol 4: Determination of anions Basic Protocol 5: Determination of elements Basic Protocol 6: Determination of total protein Basic Protocol 7: Determination of non-targeted and targeted secondary metabolites Basic Protocol 8: Determination of carotenoids, chlorophylls, and tocopherol Basic Protocol 9: Determination of starch quality.

Development of a compartmentalized model for insight into the structured metabolic pathway of carbon metabolism in cassava leaves

In silico metabolic modeling has enabled systematic study of complicated metabolic processes underlying phenotypes of organisms. Modeling of plant metabolism is often hampered by the network complexity and lack of adequate knowledge. The existing metabolic networks of cassava only cover broad metabolism and are not compartmentalized to truly represent metabolism in photosynthetic tissues. To address the aforementioned limitations and develop a robust metabolic network, physiological and genomic data derived from cassava and leaf models of Arabidopsis and rice were to extend the scope of the existing model. The proposed compartmentalized network of metabolism in photosynthetic tissues of cassava, ph-MeRecon (photosynthetic-Manihot esculenta Metabolic Pathway Reconstruction) was developed based on the information resulting of the comparative study of multiple model plants and cassava genome. The ph-MeRecon covers primary carbon metabolism and comprises 461 metabolites, 550 reactions, and 1,037 metabolic genes. Enzymatic genes on the network were validated using RNA-expression data, and the reactions and pathways were compartmentalized into cytoplasm, chloroplast, mitochondria, and peroxisome. To ensure network connectivity, metabolic gaps were filled using gap reactions obtained from literature and metabolic pathway omnibus. In addition, information on plant physiology, including photosynthetic light-dependent reactions, carboxylase and oxygenase activity of RuBisCO enzyme, and phosphoenolpyruvate carboxylase enzyme activity was incorporated into ph-MeRecon to mimic cellular metabolism in cassava leaves. Thus, ph-MeRecon offers a multi-level platform for system analysis of cellular mechanisms underlying phenotypes of interest in cassava. The ph-MeRecon metabolic model is available at http://bml.sbi.kmutt.ac.th/ph-MeRecon/.

Histological Studies of Cassava (Manihot esculenta Crantz) Root Infection Using Aspergillus niger Rhizopus stolonifer, Aspergillus niger, Aspergillus flavus, Botryodiplodia theobromae and Trichoderma viride

Histological studies were carried out to investigate the impact of some pathogenic organisms on infected cassava root after different periods of incubation. Cassava roots inoculated with Aspergillus niger, Rhizopus stolonifer, Aspergillus flavus, and Trichoderma viride showed progressive depletion of the starch grains in the cells and tissue maceration of the cassava root as the period of incubation increased. Cassava root inoculated with A. flavus showed tissue necrosis and slight fragmentation of the starch grains whereas, cassava tissues infected with A. niger showed tissue necrosis and general maceration of the starch grains within the tissue thereby creating empty spaces within the cells with no clear arrangement of the starch grains. Inoculation of the cassava tissues with R. stolonifer resulted in rapid loss of the starch grains and erosion of surrounding cell boundaries. Cassava tissue colonized by T. viride showed tissue necrosis and maceration of the starch grains. Healthy (uninoculated) cassava root tissue showed intact cells packed with large starch grains suggesting that in the host-parasite relationship, the pathogens established themselves inter and intracellularly in the parenchymatous cells of the cortex of the cassava root clearing them of starch grains, and resulting to poor yield and nutritional value of cassava and also economic loss.

Application of fluorescent and UV-Vis detection methods to profile antimicrobial activity of cassava tissues for an efficient Agrobacterium-mediated transformation.

The majority of tissue culture and transformation studies in cassava (Manihot esculenta Crantz) focus on the modification of conditions in order to establish a better protocol. Although this is a standard approach for making progress in genetic transformation technology for a target plant variety, serious difficulty still remains due to the limited applicability and adaptability of the given protocol. In the present study, we aim to develop a new concept that focuses on the development of simple but adaptable genetic transformation technology in cassava. In order to establish an efficient transformation protocol, two local edible cassava varieties, R-type, with a broad leaf with reddish petiole, and S-type, with a thin leaf with shiny greenish petiole, were obtained from Okinawa, Japan. Three detection methods, i.e., fluorescence microscopy, thin-layer chromatography (TLC) with detection under an ultraviolet (UV) illumination (254 nm) and light emitting diode (LED) illuminations (365 nm and 500 nm) without any staining, and a spectrum scanning (250-700 nm) by a microplate reader system were employed to identify a series of unique features of the petioles and leaves. Antimicrobial activity of methanol extracts from the tissues was also assayed. We succeeded in the transient expression of the GUS gene using cassava leaves and also established stable introduction of the GUS gene into three organogenic cassava calli by adapting Agrobacterium-mediated transformation. With all the findings, we have identified a flexible tool to create a better match between explants and Agrobacterium strains.

An optimized isolation protocol yields high-quality RNA from cassava tissues (Manihot esculenta Crantz).

We developed and modified a precise, rapid, and reproducible protocol isolating high‐quality RNA from tissues of multiple varieties of cassava plants (Manihot esculenta Crantz). The resulting method is suitable for use in mini, midi, and maxi preparations and rapidly achieves high total RNA yields (170–600 μg·g−1) using low‐cost chemicals and consumables and with minimal contamination from polysaccharides, polyphenols, proteins, and other secondary metabolites. In particular, A 260 : A 280 ratios were > 2.0 for RNA from various tissues, and all of the present RNA samples yielded ribosomal integrity number values of greater than six. The resulting high purity and quality of isolated RNA will facilitate downstream applications (quantitative reverse transcriptase‐polymerase chain reaction or RNA sequencing) in cassava molecular breeding.

Colletotrichum species associated with cassava anthracnose in China

AbstractAnthracnose caused by Colletotrichum (CAD) is an economically important disease of cassava; however, research on its species diversity and geographical distribution of Colletotrichum in China remains limited. In this study, we investigated the phylogenetic diversity of Colletotrichum isolates associated with symptomatic leaf tissues of cassava from Guangxi and Yunnan provinces in China and aimed to confirm their identification using advanced techniques. Based on multi‐locus phylogenetic analyses and phenotypic characteristics, we identified four species from the isolates, comprising C. plurivorum, C. karstii, C. fructicola and C. siamense, and we found variation in degrees of virulence among the species and between cassava varieties. Our results are the first to report these species from cassava, and they provide a basis for the development of management strategies for CAD in cassava.

Methodologies for selecting cassava with resistance to dry and black root rot under controlled conditions

ABSTRACT Cassava root rot diseases such as dry and black root rot are listed among the major threats, since its affect the main product (tuberous roots), causing a progressive decline in yield and affecting subsequent crop cycles, being the use of resistant varieties the most reliable control measure on field level. The objective of this study was to identify inoculation methods for the early evaluation of genotypes, considering the level of resistance to dry (DRR) and black (BRR) root rot diseases. Different methodologies and plant tissues were evaluated, based on the immersion of cassava tissues (roots and stem cuttings), soil infestation, and inoculation of detached tissues (leaves, tuberous roots and stem cuttings). The following parameters were evaluated for inoculations based on tissue immersion: disease index (ω); aerial part weight (g); fresh weight of the roots (g); and volume (cm3). For the inoculations on detached tissues, the percentage of lesioned area was determined. Immersion methods for roots and lesioned stems did not show typical symptoms of DRR and BRR during the two-month evaluation period. The soil infestation method did not differ from the stem immersion method as to the reduction of aerial part weight and the disease index, whereas both can be recommended for resistance selection trials. There was a positive correlation between the BRR and DRR data for the stem inoculation (r = 0.94, p = 0.001) and for DRR in the peel and root pulp (r = 0.73, p = 0.05). Therefore, the resistance within each tissue is apparently independent and should be compared with the behavior of the genotypes in the field.

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.

Interactive effects of temperature and drought on cassava growth and toxicity: implications for food security?

Cassava is an important dietary component for over 1 billion people, and its ability to yield under drought has led to it being promoted as an important crop for food security under climate change. Despite its known photosynthetic plasticity in response to temperature, little is known about how temperature affects plant toxicity or about interactions between temperature and drought, which is important because cassava tissues contain high levels of toxic cyanogenic glucosides, a major health and food safety concern. In a controlled glasshouse experiment, plants were grown at 2 daytime temperatures (23°C and 34°C), and either well-watered or subject to a 1month drought prior to harvest at 6months. The objective was to determine the separate and interactive effects of temperature and drought on growth and toxicity. Both temperature and drought affected cassava physiology and chemistry. While temperature alone drove differences in plant height and above-ground biomass, drought and temperature×drought interactions most affected tuber yield, as well as foliar and tuber chemistry, including C:N, nitrogen and cyanide potential (CNp; total cyanide released from cyanogenic glucosides). Conditions that most stimulated growth and yield (well-watered×high temperature) effected a reduction in tuber toxicity, whereas drought inhibited growth and yield, and was associated with increased foliar and tuber toxicity. The magnitude of drought effects on tuber yield and toxicity were greater at high temperature; thus, increases in tuber CNp were not merely a consequence of reduced tuber biomass. Findings confirm that cassava is adaptable to forecast temperature increases, particularly in areas of adequate or increasing rainfall; however, in regions forecast for increased incidence of drought, the effects of drought on both food quality (tuber toxicity) and yield are a greater threat to future food security and indicate an increasing necessity for processing of cassava to reduce toxicity.

Purification and Characterization of a Polygalacturonase Produced by Wickerhamomyces anomalus

The aim of this work was to study the purification and physicochemical properties of an endo-polygalacturonase (PG) produced by Wickerhamomyces anomalus isolated from the citrus fruit peels. The enzyme was purified to homogeneity from the culture filtrate of W. anomalus grown on the yeast nitrogen base medium with glucose as carbon and energy source and citrus pectin as inductor. After anion-exchange chromatography and gel filtration chromatography, PG activity was eluted as a single peak, yielding 21% of the original activity. After dialysis and cation-exchange chromatography, only one fraction with PG activity was obtained, recovering 56% of initial enzyme activity and 1.3-fold increase in specific activity. The molecular weight of the enzyme was estimated as 43 kDa by the SDS-PAGE. The enzyme exhibited maximal activity at pH 4.2 and was stable over a pH range from 3.5 to 6.0 and up to 49ºC for 10 h. The Vmax and Km values with polygalacturonic acid as substrate were 0.26 mmol/L.min and 0.173 mg/mL, respectively. Cations such as Cu+2, Fe+3, Mg+2, Mn+2 and Zn+2 did not show any significant effect on PG activity but K+ and Ca+2 reduced it. The purified PG was able to macerate cassava tissues.

Purification and Characterization of a Polygalacturonase Produced by Wickerhamomyces anomalus

The aim of this work was to study the purification and physicochemical properties of an endo-polygalacturonase (PG) produced by Wickerhamomyces anomalus isolated from the citrus fruit peels. The enzyme was purified to homogeneity from the culture filtrate of W. anomalus grown on the yeast nitrogen base medium with glucose as carbon and energy source and citrus pectin as inductor. After anion-exchange chromatography and gel filtration chromatography, PG activity was eluted as a single peak, yielding 21% of the original activity. After dialysis and cation-exchange chromatography, only one fraction with PG activity was obtained, recovering 56% of initial enzyme activity and 1.3-fold increase in specific activity. The molecular weight of the enzyme was estimated as 43 kDa by the SDS-PAGE. The enzyme exhibited maximal activity at pH 4.2 and was stable over a pH range from 3.5 to 6.0 and up to 49ºC for 10 h. The Vmax and Km values with polygalacturonic acid as substrate were 0.26 mmol/L.min and 0.173 mg/mL, respectively. Cations such as Cu+2, Fe+3, Mg+2, Mn+2 and Zn+2 did not show any significant effect on PG activity but K+ and Ca+2 reduced it. The purified PG was able to macerate cassava tissues.

Expression Profiling of Genes Associated with Cyanogenesis in Three Cassava Cultivars Containing Varying Levels of Toxic Cyanogens

Cyanogenic glycosides, linamarin and lotaustralin, are presents in all tissues of Cassava (Manihot esculenta Crantz) except seeds and function as a deterrent for herbivores as well as the translocable form of reduced nitrogen. The genes involved in the cyanogenic pathway [CYP79D1/D2 (EC 1.14.13), linamarase (EC 3.2.1.21), α-hydroxynitrile lyase (HNL, acetone-cyanohydrin lyase. EC 4.1.2.37) and b-cyanoalanine synthase (β-CAS. EC 4.4.1.9] have been identified and partially characterized. Our objective was to identify the differential expression pattern of these genes in leaves and roots of three cassava cultivars with varying levels of cyanogenic glucosides. The results show that the differential ex- pression of the genes between leaves and roots is consistent with leaves being the primary site of synthesize of cyano- genic glucosides, which are then translocated to the roots. In addition, the varietal difference for cyanogenic glucoside levels could be explained in part by the combinatorial effort of the synthesis in the leaves and the linamarase catabolic step in the roots. Cluster analysis suggests a coordinated expression between CYP79D1/D2 and β-CAS genes as well as linamarase and HNL genes, which is in agreement with the spatial separation within a cell of the site of linamarin syn- thesis (vacuolar) and its breakdown to cyanide (cell wall). Furthermore, cluster analysis for cultivar classification using its gene expression profile match with the reported cyanide levels comparatively for the three cultivars. This is the first study that evaluates the transcriptional activities of the genes involved in the cyanogenic glycoside metabolism using a systematic approach.

Variations in the Chemical Composition of Cassava (Manihot esculenta Crantz) Leaves and Roots As Affected by Genotypic and Environmental Variation

The purpose of this study was to assess the quality of cassava cultivars, in terms of cyanogenic potential and composition of macro- and micronutrients, sampled from different locations in rural Mozambique. Total cyanide concentrations in fresh cassava tissues were measured using portable cyanide testing kits, and elemental nutrients were later analyzed from dried plant tissue. Variation in cyanogenic potential and nutrient composition occurred both among cultivars and across locations. The majority of cultivars contained >100 ppm total cyanide, fresh weight, and are therefore considered to be dangerously poisonous unless adequately processed before consumption. Leaf cyanogenic and nutrient content varied with plant water status, estimated using carbon isotope discrimination (δ(13)C). The colonization of roots of all cultivars by arbuscular mycorrhizal fungi was also quantified and found to be high, indicating that mycorrhizas could play a key role in plant nutrient acquisition in these low-input farming systems.

Bioinformatic identification of cassava miRNAs differentially expressed in response to infection by Xanthomonas axonopodis pv. manihotis.

BackgroundmicroRNAs (miRNAs) are short RNA molecules that control gene expression by silencing complementary mRNA. They play a crucial role in stress response in plants, including biotic stress. Some miRNAs are known to respond to bacterial infection in Arabidopsis thaliana but it is currently unknown whether these responses are conserved in other plants and whether novel species-specific miRNAs could have a role in defense.ResultsThis work addresses the role of miRNAs in the Manihot esculenta (cassava)-Xanthomonas axonopodis pv. manihotis (Xam) interaction. Next-generation sequencing was used for analyzing small RNA libraries from cassava tissue infected and non-infected with Xam. A full repertoire of cassava miRNAs was characterized, which included 56 conserved families and 12 novel cassava-specific families. Endogenous targets were predicted in the cassava genome for many miRNA families. Some miRNA families' expression was increased in response to bacterial infection, including miRNAs known to mediate defense by targeting auxin-responding factors as well as some cassava-specific miRNAs. Some bacteria-repressed miRNAs included families involved in copper regulation as well as families targeting disease resistance genes. Putative transcription factor binding sites (TFBS) were identified in the MIRNA genes promoter region and compared to promoter regions in miRNA target genes and protein coding genes, revealing differences between MIRNA gene transcriptional regulation and other genes.ConclusionsTaken together these results suggest that miRNAs in cassava play a role in defense against Xam, and that the mechanism is similar to what's known in Arabidopsis and involves some of the same families.