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

BACKGROUND:Whiteflies 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. RESULTS:The 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. CONCLUSIONS:This 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

Recent Biotechnological Advances in the Improvement of Cassava

Cassava (Manihot esculenta Crantz) is the fourth most important source of carbohydrates for human consumption in the tropics and thus occupies a uniquely important position as a food security crop for smallholder farmers. Consequently, cassava improvement is of high priority to most national agricultural research institutions in the tropics. With advances in functional genomics and genome editing approaches in this post genomics era, there are unprecedented opportunities and potential to accelerate the improvement of this important crop. These new technologies will need to be directed toward addressing major cassava production constraints, notably virus resistance, protein content, tolerance to drought and reduction of hydrogen cyanide content. Here, we discuss the important role novel functional genomics and genome editing technologies have and will continue to play in cassava improvement efforts. These approaches, including artificial miRNA (amiRNA), trans-acting small interfering RNA (tasiRNA), clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9), and Targeting Induced Local Lesions IN Genomes (TILLING), have been shown to be effective in addressing major crop production constraints. In addition to reviewing specific applications of these technologies in cassava improvement, this chapter discusses specific examples being deployed in the amelioration of cassava or of other crops that could be applied to cassava in future

The role of small RNAs in susceptibility and tolerance to cassava mosaic disease

A dissertation presented by Sarah Jane Rogans to The Faculty of Science, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Molecular and Cell Biology. 2016Cassava (Manihot esculenta, Crantz) is considered to be an important food security crop consumed by over a billion peoples globally, many who subsist on it. Cassava mosaic disease (CMD) is one of the main biotic and economically important constraints to cassava cultivation in sub-Saharan Africa. Geminiviruses are the casual agents of CMD and cause disease to many staple food and cash crops of great economic importance worldwide. There are currently 11 species of Begomoviruses that belong to the Geminiviridae family. South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite (DNA A and DNA B components) begomovirus belonging to the family Geminiviridae, and is one of the causal agents of cassava mosaic disease (CMD) endemic to southern Africa. Various strategies to control CMD are currently being investigated, one of which is cis-genics, which involves manipulation of endogenous host genes to combat viral pathogens. In order to achieve this, it is imperative to elucidate molecular mechanisms involved in host-virus interactions. Endogenous small RNAs (sRNAs), including microRNAs (miRNAs), have been found associated with gene regulatory mechanisms in response to virus infection. Amongst the non-coding host sRNAs targeting viruses are small interfering RNAs (siRNAs) associated with posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS), which are involved in the host RNA silencing pathway. The RNA silencing pathway is a highly conserved basal immunity pathway involved in host defence against plant viruses. The aim of this study was to identify siRNAs and miRNAs associated with gene regulatory mechanism in response to SACMV infection and to determine if they a play a role in the susceptible or recovery phenotype observed in SACMV tolerant cassava landrace TME3 or T200, respectively. Furthermore, virus-derived siRNA (vsRNA) populations targeting the DNA A and B components of SACMV were also investigated. MicroRNAs (miRNAs) are an important class of endogenous non-coding single-stranded small RNAs (21-24 nt in length), which serve as post-transcriptional negative regulators of gene expression in plants. Despite the economic importance of Manihot esculenta Crantz (cassava) only 153 putative cassava miRNAs (from multiple germplasm) are available to date in miRBase (V.21). Therefore, both conserved and novel miRNAs needed to be identified in cassava before we could determine what association they had with SACMV infection. In this part of the study, mature sequences of all known plant miRNAs were used as a query for homologous searches against cassava EST and GSS databases, and additional identification of novel and conserved miRNAs were gleaned from next generation sequencing (NGS) of two cassava landraces (T200 from southern Africa and TME3 from West Africa) at three different growth stages post explant transplantation and acclimatization. EST and GSS derived data revealed 259 and 32 conserved miRNAs in cassava, and one of the miRNA families (miR2118) from previous studies has not been reported in cassava. NGS data collectively displayed expression of 289 conserved miRNAs in leaf tissue, of which 230 had not been reported previously. Of the 289 conserved miRNAs identified in T200 and TME3, 208 were isomiRs. Thirty-nine novel cassava-specific miRNAs of low abundance, belonging to 29 families, were identified. Thirty-eight (98.6%) of the putative new miRNAs identified by NGS have not been previously reported in cassava. Several miRNA targets were identified in T200 and TME3, highlighting differential temporal miRNA expression between the two cassava landraces. This study contributes to the expanding knowledge base of the micronome of this important crop. MicroRNAs play a crucial role in stress response in plants, including biotic stress caused by viral infection. Viruses however can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of miRNAs. This study aimed to understand the regulation of miRNAs in tolerant (TME3) and susceptible (T200) cassava landraces infected with SACMV. Next-generation sequencing was used for analysing small RNA libraries from infected and mock-inoculated cassava leaf tissue collected at 12, 32 and 67 dpi (days post-inoculation). The total number of differentially expressed miRNAs (normalized against mock-inoculated samples) across all three time points was 204 and 209 miRNAs, in TME3 and T200 infected plants, respectively, but the patterns of log2fold changes in miRNA families over the course of infection differed between the two landraces. A high number were significantly altered at 32 dpi when T200 and TME3 plants showed severe symptoms. Notably, in T200 69% and 28 (100%) of miRNA families were upregulated at 12 and 32 dpi, respectively. In contrast, TME3 showed an early pre-symptomatic response at 12 dpi where a high number (87%) of miRNAs showed a significant log2fold downregulation. Endogenous targets were predicted in the cassava genome for many of the identified miRNA families including transcription factors, disease resistance (R)-genes and transposable elements. Interestingly, some of the miRNA families (miR162, miR168 and miR403) that were significantly affected in both T200 and TME3 upon SACMV infection were shown to target proteins (DCL1, AGO1 and AGO2) that play important roles in the RNA silencing pathway. From results, we suggest that the early (12 dpi) miRNA response to SACMV in TME3 appears to involve PTGS-associated AGO1, DCL2 and a cohort of R genes belonging to the miR395 family which may prime the plant for tolerance and recovery downstream, while in T200, SACMV suppresses AGO1, AGO2 (at 32 and 67 dpi), and DCL2 (32 dpi) mediated RNA silencing, leading to severe persistent disease symptoms. This study provides insights into miRNA-mediated SACMV cassava interactions and may provide novel targets for control strategies aimed at developing CMD-resistance cassava varieties Endogenous small RNAs (sRNAs) associated with gene regulatory mechanisms respond to virus infection, and virus-derived small interfering RNAs (vsRNAs) have been implicated in recovery or symptom remission in some geminivirus-host interactions. Transcriptional gene silencing (TGS) (24 nt vsRNAs) and post transcriptional gene silencing (PTGS) (21-23 nt vsRNAs) have been associated with geminivirus intergenic (IR) and coding regions, respectively. In this Illumina deep sequencing study, we compared for the first time, the small RNA response to South African cassava mosaic virus (SACMV) of cassava landrace TME3 which shows a recovery and tolerant phenotype, and T200, a highly susceptible landrace. Interestingly, different patterns in the percentage of SACMV-induced normalized total endogenous sRNA reads were observed between T200 and TME3. Notably, in T200 there was a significant increase in 21 nt sRNAs during the early pre-symptomatic response (12 dpi) to SACMV compared to mock, while in TME3, the 22 nt size class increased significantly at 32 dpi. While vsRNAs of 21 to 24 nt size classes covered the entire SACMV DNA- A and DNA-B genome components in T200 and TME3, vsRNA population counts were significantly lower at 32 (symptomatic stage) and 67 dpi in tolerant TME3 compared with T200 (non-recovery). It is suggested that the high accumulation of primary vsRNAs, which correlated with high virus titres and severe symptoms in susceptible T200, may be due to failure to target SACMV-derived mRNA. In contrast, in TME3 low vsRNA counts may represent efficient PTGS of viral mRNA, leading to a depletion/sequestration of vsRNA populations, supporting a role for PTGS in tolerance/recovery in TME3. Notably, in TME3 at recovery (67 dpi) the percentage (expressed as a percentage of total vsRNA counts) of redundant and non-redundant (unique) 24 nt vsRNAs increased significantly. Since methylation of the SACMV genome was not detected by bisulfite sequencing, and vsRNA counts targeting the IR (where the promoters reside) were very low in both the tolerant or susceptible landraces, we conclude that 24 nt vsRNA-mediated RNA directed genome methylation does not play a central role in disease phenotype in these landraces, notwithstanding recognition for a possible role in histone modification in TME3. This work represents an important step toward understanding variable roles of sRNAs in different cassava genotype-geminivirus interactions. Also, by comparing the differences between a tolerant and susceptible host the aim is to achieve better understanding of the effect of pathogens on host sRNAome, an area that is deserving of me attention in plant systems. The expectation is that these findings presented in the PhD will contribute to the long-term goals of devising new methods of disease control against SACMV and understanding the complex interconnected mechanisms involved in virus-host interactome.LG201

Endogenous small-noncoding RNAs and their roles in chilling response and stress acclimation in Cassava

BACKGROUND: Small noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs) are key gene regulators in eukaryotes, playing critical roles in plant development and stress tolerance. Trans-acting siRNAs (ta-siRNAs), which are secondary siRNAs triggered by miRNAs, and siRNAs from natural antisense transcripts (nat-siRNAs) are two well-studied classes of endo-siRNAs. RESULTS: In order to understand sncRNAs’ roles in plant chilling response and stress acclimation, we performed a comprehensive study of miRNAs and endo-siRNAs in Cassava (Manihot esculenta), a major source of food for the world populations in tropical regions. Combining Next-Generation sequencing and computational and experimental analyses, we profiled and characterized sncRNA species and mRNA genes from the plants that experienced severe and moderate chilling stresses, that underwent further severe chilling stress after chilling acclimation at moderate stress, and that grew under the normal condition. We also included castor bean (Ricinus communis) in our study to understand conservation of sncRNAs. In addition to known miRNAs, we identified 32 (22 and 10) novel miRNAs as well as 47 (26 and 21) putative secondary siRNA-yielding and 8 (7 and 1) nat-siRNA-yielding candidate loci in Cassava and castor bean, respectively. Among the expressed sncRNAs, 114 miRNAs, 12 ta-siRNAs and 2 nat-siRNAs showed significant expression changes under chilling stresses. CONCLUSION: Systematic and computational analysis of microRNAome and experimental validation collectively showed that miRNAs, ta-siRNAs, and possibly nat-siRNAs play important roles in chilling response and chilling acclimation in Cassava by regulating stress-related pathways, e.g. Auxin signal transduction. The conservation of these sncRNA might shed lights on the role of sncRNA-mediated pathways affected by chilling stress and stress acclimation in Euphorbiaceous plants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-634) contains supplementary material, which is available to authorized users

Elucidation of the role of NOA1 and myosins in host response to infection by SACMV

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy in Science at the School of Molecular and Cell Biology, 2017Different host genes playing a role in replication, transcription and movement of geminiviruses have been identified, allowing a better understanding of host response during infection. The cytoskeletal protein myosin has been shown to associate with RNA viruses movement protein and mediate its movement, however no geminivirus association with myosin has been established. Arabidopsis thaliana nitric oxide associated protein 1 (AtNOA1), once thought to be an enzyme involved in a nitric oxide (NO) production, has been reported to be differentially regulated in response to biotic and abiotic stress. In this study we sought to identify the role that myosins and NOA1 play in the development of disease by south african cassava mosaic virus (SACMV). Using a bioinformatics approach, 24 myosin transcripts were identified in Nicotiana benthamiana, and phylogeny analysis revealed that seven were class VIII myosins and 17 class XI. Five myosins silencing constructs M15.1 (transcript Niben101Scf11288g00015.1), MYOSIN XI-F (M11.F), MYOSIN XI-K (M11.K), MYOSIN XI-2 (M11.2) and MYOSIN VIII.B were selected for silencing using a virus induced gene silencing (VIGS) approach with SACMV and TRV-VIGS vectors. At 14 days post inoculation (dpi), both SACMV and TRV-VIGS vectors successfully silenced myosins with SACMV-VIGS silencing all five and TRV-VIGS silencing all but M11. F. At 28 dpi, SACMV-VIGS induced silencing of myosin of only two myosins and TRV-VIGS three. TRV-VIGS was found to be more efficient at silencing as the suppression of myosin induced by TRV-VIGS was stronger than that of SACMV-VIGS. To assess the effect of myosin silencing on SACMV infectivity in a separate experiment, 7 dpi of silencing, N. benthamiana plants were challenged with SACMV and reduction of myosin expression was assessed as well as viral accumulation. TRV-VIGS did not induce any silencing of myosin at 14 dpi, and at 28 dpi, the expression of M11.K and M11.F were silenced. SACMV-VIGS induced silencing of M11.F at both 14 and 28 dpi. In TRV-VIGS silenced M11.K, viral load at 28 dpi was not lower than the control, however the fold increase in viral load at 28 dpi compared to 14 dpi was 3-fold (p value 0.03) for M11.K silenced TRV-VIGS plants and 86-fold for the control 6-fold for the M11.K suggesting that silencing of M11.K decreases the spread of SACMV. In TRV-VIGS silenced M11.K, viral load at 28 dpi was lower than the control (9-fold p value 0.03) and the increase in viral load at 28 dpi compared to 14 dpi was insignificant, suggesting that spreading of SACMV was also hampered. The reduction in myosin M11.F expression induced iv by SACMV-VIGS resulted in an increase in viral load compared to the control. We hypothesise that the increase in viral load observed in M11.F silenced plants induced by SACMV-VIGS is due to the perceived resistance of SACMV-VIGS control (SACMV-challenged no silencing construct) to SACMV-challenge, and therefore results from the SACMV-VIGS study were inconclusive. From the TRV-VIGS study however, we have identified two candidate myosins in N. benthamiana myosin XI-K and myosin XI-F as potential interactor of SACMV during infectivity. Further research into their role in the development of SACMV disease is warranted. Nitric oxide associated 1 (NOA1) in plants is a cyclic GTPase involved in protein translation in the chloroplast and has been indirectly linked to nitric oxide (NO) accumulation. To understand the role played by NOA1 in response to (SACMV) infection, a bioinformatics approach was used to identify NOA1 homologues in cassava T200. Using the cassava genome data on Phytozome, a putative NOA1 namely cassava 4.1_007735m, was identified. Based on its protein sequence, cassava4.1_007735m shared a 69.6% similarity to Arabidopsis NOA1 (AtNOA1). The expression of cassava4.1_007735.m (MeNOA1) and N. benthamiana NOA1 (NbNOA1) and the accumulation of NO in leaf samples was compared between SACMV-infected and non-infected at early infection stage (14 dpi for N. benthamiana and 28 dpi for cassava T200) and full systemic stage (28 dpi for N. benthamiana and 56 dpi for cassava T200). Real-time PCR was used to measure SACMV viral load which increased significantly by 2-fold (p value 0.05) from 14 to 28 dpi for N. benthamiana and 8-fold from 28 to 56 dpi in cassava T200 (p value 0.04) as chlorosis and symptom severity concomitantly progressed. At 14 and 28 dpi, NbNOA1 expression was significantly lower than mock inoculated plants (2-fold lower at 14 dpi, p value 0.01 and 4 fold lower at 28, (p value 0.00) and the abundance of NO in infected N. benthamiana leaf tissue was 10% lower at 14 dpi and 40% lower at 28 dpi when compared to mock inoculated. In cassava T200, MeNOA1 expression was unchanged at 28 dpi and NO levels were decreased by 40% and at 56 dpi, MeNOA1 expression was 4-fold lower and NO accumulation was 37 % higher than that of mock inoculated leaf tissue. At 28 dpi for N. benthamiana and 56 for cassava T200, the decrease in NOA1 expression was accompanied by chloroplast dysfunction, evident from the significant reduction in chlorophylls a and b and carotenoids in SACMV-infected leaf samples. Furthermore, the expression of v chloroplast translation factors (chloroplast RNA binding, chloroplast elongation factor G, translation initiation factor 3-2, plastid-specific ribosomal protein 6 and) were found to be repressed in infected N. benthamiana and infected cassava T200 relative to mock inoculated plants. GC-MS analysis showed a decrease in fumarate and an increase in glucose in SACMV-infected N. benthamiana in comparison to mock samples suggesting a decrease in carbon stores. Collectively, these results provide evidence that in response to SACMV infection in N. benthamiana, decrease in photopigment and carbon stores, accompanied by an increase in glucose and decrease in fumarate, lead to a decline in NbNOA1 and NO levels. This is manifested by suppressed translation factors, and disruption of the chloroplast, resulting in chlorotic disease symptoms. In cassava T200 however, the link could not be established as the level of glucose was not significantly decreased and fumaric acid was not detected and although the concomitant decrease in the expression of MeNOA1 and chloroplast translation factors indicate dysfunction of the chloroplast, the link between MeNOA1 expression, carbon store, NO and chloroplast activity could not be established.XL201

siRomics for universal diagnostics of plant viral disease and virus diversity studies

Traditional methods of viral diagnostics using specific antibodies and PCR often fail to identify a viral pathogen. In our EU Marie-Curie IDP bridges project, we used an alternative novel approach called siRomics which allows not only to detect the virus but also to de novo reconstruct a complete consensus master genome in the viral quasispecies population. The main plant antiviral defense system is based on RNA silencing mediated by small RNAs. In plants infected with DNA and RNA viruses, host Dicer enzymes generate 21-24 nucleotide (nt) viral small interfering RNAs (siRNAs) that restrict virus replication and systemic spread. Growing evidence indicates that viral siRNAs are derived from the entire genome sequence of RNA and DNA viruses and accumulate at high levels. Hence it appears feasible to reconstruct a complete viral genome simply from viral siRNA species. Current bioinformatics algorithms enable de novo assembly of genomes and transcriptomes from short sequencing reads. In the past years, the siRomics pipeline, developed by Seguin et al. (2014b) in model plants, was further applied in crop plants (Seguin et al. 2014b, 2016, Rajeswaran et al. 2014a, 2014b, Fuentes et al. 2016). Thus, our siRomics approach has the potential for universal diagnostics of plant virus disease and de novo reconstruction of viral genomes in mixed infections. In this study we applied siRomics for virus detection and virome reconstruction in several case studies of economically-important viral diseases in Switzerland. In naturally-infected Solanum tuberosum (potato), one case study revealed a virome comprising Potato virus Y (genus Potyvirus) and Potato virus X (genus Potexvirus), which was reconstructed by de novo assembling separate genome-size sRNA contigs. Another case study revealed a virome comprising NTN and O strains of Potato virus Y, whose sRNAs assembled in chimeric contigs which could be disentangled on the basis of reference genome sequences. Both viromes were stable in vegetative potato progeny. In a cross-protection trial of Solanum lycopersicum (tomato), the supposedly protective mild strain CH2 of Pepino mosaic virus (Potexvirus) was tested for protection against the strain LP of the same virus. Reciprocal mechanical inoculations eventually resulted in co-infection of all individual plants with CH2 and LP strains, reconstructed as separate sRNA contigs. LP invasions into CH2-preinfected plants and vice versa were accompanied by alterations of consensus genome sequences in viral quasispecies, indicating a potential risk of cross-protection measures. Additionally, the study also revealed, by reconstruction from sRNAs, the presence of the mechanically non- transmissible Southern tomato virus (Amalgavirus) in some plants. Our in-depth analysis of sRNA sizes, 5'-nucleotide frequencies and hotspot maps revealed similarities in sRNA- generating mechanisms in potato and tomato, differential silencing responses to virome components and potential for sRNA-directed cross-targeting between viral strains which could not, however, prevent the formation of stable viromes. Furthermore, by siRomics we characterized the virome present in cultivated and non-cultivated perennial plants including grapevine, cherry, fig, privet and larch. As expected, grapevine samples showed a complex virome, including viroids, in particular Grapevine Fanleaf virus, Grapevine virus A, Grapevine leafroll associated virus, Yellow speckle viroid 1, Yellow speckle viroid 2, Hopstunt viroid and Australian grapevine viroid. In cherry trees affected by little cherry disease, we confirmed that the presence of two Little cherry virus (1 and 2, respectively) in one of the samples, induces more severe symptoms compared with the sample where only Little cherry virus 1 was present. In a fig tree exhibiting virus-like symptoms coming from a private garden, new isolates of Fig mosaic virus and Fig Badnavirus-1 were identified and reconstructed. In the forest bush plant privet (Ligustrum vulgare) showing yellow mosaic disease, a novel virus distantly related to Barley yellow strip virus and Lychnis ringspot virus was identified, fully reconstructed and named Ligustrum mosaic virus. Our work combined multi-disciplinary approaches ranging from advanced molecular methods of next generation sequencing to sophisticated bioinformatics algorithms for virus genome reconstruction. The results of our study are informative for further understanding the mechanisms of RNA silencing-based antiviral defense, which would contribute to basic research in the field of plant-pathogen interaction, and for developing novel strategies of virus control, which could potentially be implemented in the future in Swiss agriculture though our recommendations to the policy makers. In modern agriculture, horticulture and (bio-) farming, it becomes critical to assess the risk of emerging plant infections and to control the spread of plant viral diseases

Transference of RXam2 and Bs2 genes to confer resistance against cassava bacterial blight (CBB)

Abstract. Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is a major disease in all regions where cassava is cultivated. To generate broad spectrum and durable CBB resistance it is necessary pyramiding several R genes. We have identified two candidate resistance genes, named RXam1 and RXam2 which colocalize with QTLs associated to resistance against Xam. RXam1 codes for a RLK (Receptor-Like Kinase) protein. We generated several transgenic cassava lines overexpressing RXam1. In vitro plants of three lines showed reduced symptoms and reduced bacterial growth after Xam infection with strain CIO136 compared to empty vector transgenic plants. The second cassava gene, RXam2, codes for a NB-LRR protein. Using RNAi we generated RXam2-silenced transgenic plants, which were more susceptible to several Xam strains than non-transformed plants. On the other hand, plants overexpressing RXam2 showed reduced symptoms to Xam strains. These data collectively suggest that RXam2 is a resistance gene against Xam. In addition, an autoactive version mutated in the MHD motif (NB domain) of RXam2 was generated through site directed mutagenesis and was able to generate a Hypersensitive Response (HR) by transient agroinfiltration in cassava and tobacco leaves. The autoactive version of RXam2 was cloned under a TALE1Xam-inducible promoter and transient expression in tobacco showed a strong HR when co-infiltrated with a plasmid containing the TALE1Xam gene. Several independent transgenic stable lines are being evaluated to assess TALE1Xam inducibility. Finally, several cassava transgenic plants overexpressing Bs2 from pepper were obtained and showed constitutive, typical immune responses.Doctorad