First report of Cassava Bacterial Blight caused by Xanthomonas phaseoli pv. manihotis in the Amazonian forest of Ecuador.

Xanthomonas phaseoli pv. manihotis (Xpm) is a plant pathogenic bacterium known as the causal agent of cassava bacterial blight (CBB). CBB is the most limiting bacterial disease affecting cassava (Manihot esculenta Crantz), characterized by diverse symptoms including angular water-soaked leaf lesions, blight, wilting, stem exudates, stem cankers and dieback. CBB has been reported in most cassava-growing regions around the world, and, under conducive conditions, crop yield losses can reach up to 100% (Zárate-Chaves et al. 2021). While Xpm genetic diversity is remarkably high in South America (Bart et al. 2012) and cassava originates and was domesticated in the Amazon basin (Allem 2002), reports of CBB in the Amazonian region are missing. To fill this gap, in October 2018 we surveyed for CBB symptoms in cassava fields of the Orellana Province, located in the Amazon forest of the Republic of Ecuador. Adult cassava plants exhibiting typical angular, water-soaked leaf lesions were found in polyculture plots, i.e. intercrops of cassava with other species such as plantains and fruit trees (a.k.a. chakras). After surface disinfection with 5% sodium hypochlorite followed by 70% ethanol, white Xpm-like colonies were isolated from diseased leaf tissues of four plants on YPGA medium (yeast extract, 5 g/l; peptone, 5 g/l; glucose, 5 g/l; agar-agar, 15 g/l) supplemented with cephalexin (40 mg/l) and cycloheximide (50 mg/l). Pathogenicity tests were performed on peat-potted, 2-month-old cassava plants of the cultivar 60444. Bacterial suspensions were adjusted to an OD600 of 0.2 (2 × 108 CFU/ml) in sterile 10-mM MgCl2 and syringe infiltrated in fully-expanded leaves. In parallel, 20 µl of each bacterial suspension adjusted to an OD600 of 0.02 (2 × 107 CFU/ml) were inoculated on stems inside a hole previously punched with a sterile needle in the junction of the third-top petiole. Sterile 10-mM MgCl2 was used for mock inoculations in both leaves and stems, and experiments were replicated in three plants. Plants were incubated in a greenhouse at 28 ± 1°C with a 12-h photoperiod. Infiltrated leaves developed watersoaking 3 days post inoculation, while wilted leaves, stem exudates, and dieback were observed 21 days after stem inoculation. Control plants remained symptomless. White Xpm-like colonies were re-isolated from symptomatic leaves (Fig S1). One colony of each of the four Xpm isolates (before and after re-isolation) was assessed using diagnostic PCRs (Bernal-Galeano et al. 2018; Flores et al. 2019), using strain Xam668 as positive control. All four candidates were positive for both diagnostic tools. The sequences of the housekeeping genes atpD, dnaK, efp, glnA, gyrB and rpoD of our isolates were extracted from full genome sequences obtained through Oxford Nanopore Technologies (ONT) (GenBank OR288194 to OR288217) and compared to their homologs in four close Xanthomonas species and a reference Xpm strain (Table S1). The sequences of the tested strains aligned with that of Xpm CIO151 (GCA_004025275.1) (Arrieta-Ortiz et al. 2013) with nucleotide identity above 99.92% (Fig S2). The four strains were named CIX4169, CIX4170, CIX4171 and CIX4172, stored in the IRD Collection of Xanthomonas, where they are available upon request. To our knowledge, this is the first report of CBB in the Amazonian region and in Ecuador, where cassava is a central element for local culture and economy. Further surveys will be necessary to evaluate the distribution and prevalence of CBB in other ecozones of Ecuador where cassava is cultivated.

CRISPRi in Xanthomonas demonstrates functional convergence of transcription activator-like effectors in two divergent pathogens.

Functional analysis of large gene families in plant pathogens can be cumbersome using classical insertional mutagenesis. Additionally, Cas9 toxicity has limited the application of CRISPR-Cas9 for directed mutagenesis in bacteria. Here, we successfully applied a CRISPR interference strategy to investigate the cryptic role of the transcription activator-like effector (tale) multigene family in several plant-pathogenic Xanthomonas bacterial species, owing to their contribution to pathogen virulence. Single guide RNAs (sgRNAs) designed against Xanthomonas phaseoli pv manihotis tale conserved gene sequences efficiently silenced expression of all tales, with concomitant decrease in virulence and TALE-induced host gene expression. The system is readily translatable to other Xanthomonas species infecting rice, citrus, Brassica, and cassava, silencing up to 16 tales in a given strain using a single sgRNA. Complementation with plasmid-borne designer tales lacking the sgRNA-targeted sequence restored molecular and virulence phenotypes in all pathosystems. Our results evidenced that X. campestris pv campestris CN08 tales are relevant for symptom development in cauliflower. They also show that the MeSWEET10a sugar transporter is surprisingly targeted by the nonvascular cassava pathogen X. cassavae, highlighting a new example of TALE functional convergence between phylogenetically distant Xanthomonas. Overall, this novel technology provides a platform for discovery and rapid functional understanding of highly conserved gene families.

RXam2, a NLR from cassava (Manihot esculenta) contributes partially to the quantitative resistance to Xanthomonas phaseoli pv. manihotis.

KEY MESSAGE: The overexpression of RXam2, a cassava NLR (nucleotide-binding leucine-rich repeat) gene, by stable transformation and gene expression induction mediated by dTALEs, reduce cassava bacterial blight symptoms. Cassava (Manihot esculenta) is a tropical root crop affected by different pathogens including Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of cassava bacterial blight (CBB). Previous studies have reported resistance to CBB as a quantitative and polygenic character. This study sought to validate the functional role of a NLR (nucleotide-binding leucine-rich repeat) associated with a QTL to Xpm strain CIO151 called RXam2. Transgenic cassava plants overexpressing RXam2 were generated and analyzed. Plants overexpressing RXam2 showed a reduction in bacterial growth to Xpm strains CIO151, 232 and 226. In addition, designer TALEs (dTALEs) were developed to specifically bind to the RXam2 promoter region. The Xpm strain transformed with dTALEs allowed the induction of the RXam2 gene expression after inoculation in cassava plants and was associated with a diminution in CBB symptoms. These findings suggest that RXam2 contributes to the understanding of the molecular basis of quantitative disease resistance.

Cassava diseases caused by Xanthomonas phaseoli pv. manihotis and Xanthomonas cassavae.

Xanthomonas phaseoli pv. manihotis (Xpm) and X. cassavae (Xc) are two bacterial pathogens attacking cassava. Cassava bacterial blight (CBB) is a systemic disease caused by Xpm, which might have dramatic effects on plant growth and crop production. Cassava bacterial necrosis is a nonvascular disease caused by Xc with foliar symptoms similar to CBB, but its impacts on the plant vigour and the crop are limited. In this review, we describe the epidemiology and ecology of the two pathogens, the impacts and management of the diseases, and the main research achievements for each pathosystem. Because Xc data are sparse, our main focus is on Xpm and CBB.

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.

Identification of immunity related genes to study the Physalis peruviana--Fusarium oxysporum pathosystem.

The Cape gooseberry (Physalisperuviana L) is an Andean exotic fruit with high nutritional value and appealing medicinal properties. However, its cultivation faces important phytosanitary problems mainly due to pathogens like Fusarium oxysporum, Cercosporaphysalidis and Alternaria spp. Here we used the Cape gooseberry foliar transcriptome to search for proteins that encode conserved domains related to plant immunity including: NBS (Nucleotide Binding Site), CC (Coiled-Coil), TIR (Toll/Interleukin-1 Receptor). We identified 74 immunity related gene candidates in P. peruviana which have the typical resistance gene (R-gene) architecture, 17 Receptor like kinase (RLKs) candidates related to PAMP-Triggered Immunity (PTI), eight (TIR-NBS-LRR, or TNL) and nine (CC-NBS-LRR, or CNL) candidates related to Effector-Triggered Immunity (ETI) genes among others. These candidate genes were categorized by molecular function (98%), biological process (85%) and cellular component (79%) using gene ontology. Some of the most interesting predicted roles were those associated with binding and transferase activity. We designed 94 primers pairs from the 74 immunity-related genes (IRGs) to amplify the corresponding genomic regions on six genotypes that included resistant and susceptible materials. From these, we selected 17 single band amplicons and sequenced them in 14 F. oxysporum resistant and susceptible genotypes. Sequence polymorphisms were analyzed through preliminary candidate gene association, which allowed the detection of one SNP at the PpIRG-63 marker revealing a nonsynonymous mutation in the predicted LRR domain suggesting functional roles for resistance.

Tell me a tale of TALEs.

Pathogenic bacteria of the Xanthomonas and Ralstonia genus have developed resourceful strategies creating a favorable environment to multiply and colonize their host plants. One of these strategies involves the secretion and translocation of several families of effector proteins into the host cell. The transcription activator-like effector (TALE) family forms a subset of proteins involved in the direct modulation of host gene expression. TALEs include a number of tandem 34-amino acid repeats in their central part, where specific residues variable in two adjacent positions determine DNA-binding in the host genome. The specificity of this binding and its predictable nature make TALEs a revolutionary tool for gene editing, functional analysis, modification of target gene expression, and directed mutagenesis. Several examples have been reported in higher organisms as diverse as plants, Drosophila, zebrafish, mouse, and even human cells. Here, we summarize the functions of TALEs in their natural context and the biotechnological perspectives of their use.

Mapping EST-derived SSRs and ESTs involved in resistance to bacterial blight in Manihot esculenta.

Cassava (Manihot esculenta Crantz) is a major root crop widely grown in the tropics. Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is an important disease in Latin America and Africa resulting in significant losses. The preferred control method is the use of resistant genotypes. Mapping expressed sequence tags (ESTs) and determining their co-localization with quantitative trait loci (QTLs) may give additional evidence of the role of the corresponding genes in resistance or defense. Twenty-one EST-derived simple sequence repeats (SSRs) were mapped in 16 linkage groups. ESTs showing similarities with candidate resistance genes or defense genes were also mapped using strategies such as restriction fragment length polymorphisms, cleaved amplified polymorphic sequences, and allele-specific primers. In total, 10 defense-related genes and 2 bacterial artificial chromosomes (BACs) containing resistance gene candidates (RGCs) were mapped in 11 linkage groups. Two new QTLs associated with resistance to Xam strains CIO121 and CIO151 were detected in linkage groups A and U, respectively. The QTL in linkage group U explained 61.6% of the phenotypic variance and was associated with an RGC-containing BAC. No correlation was found between the new EST-derived SSRs or other mapped ESTs and the new or previously reported QTLs.

Expression and secretion of human apolipoprotein A-I in the heart.

Various studies have correlated apolipoprotein (apo) A-I, the major component high-density lipoprotein, with protection against development of cardiovascular disease. Although apoA-I expression has been previously detected in the liver and intestine, we have discovered that the human apoA-I gene is also expressed in the heart. Using transgenic (Tg) mice generated with the human apoA-I/C-III/A-IV gene cluster and Tg mice produced with just the 2.2 kb human apoA-I gene, we have detected significant levels of apoA-I expression in the heart. Furthermore, the detection of apoA-I expression in the hearts of human apoA-I Tg mice indicates that the minimal regulatory elements necessary for cardiac expression of the gene are located near its coding sequence. To determine if the apoA-I gene is also expressed in the human heart, similar analyses were performed, where apoA-I expression was found in both adult and fetal hearts. Furthermore in-depth investigation of the various regions of human and Tg mouse hearts revealed that the apoA-I mRNA was present in the ventricles and atria, but not in the aorta. In situ hybridization of Tg mouse hearts revealed that apoA-I expression was restricted to the cardiac myocyte cells. Finally, heart explants and cardiac primary culture experiments with Tg mice showed secretion of particles containing the human apoA-I protein, and metabolic labeling experiments have also detected a 28 kDa human apoA-I protein secreted from the heart. From these novel findings, new insights into the role and function of apoA-I can be extrapolated.

Identifying resistance gene analogs associated with resistances to different pathogens in common bean.

ABSTRACT A polymerase chain reaction approach using degenerate primers that targeted the conserved domains of cloned plant disease resistance genes (R genes) was used to isolate a set of 15 resistance gene analogs (RGAs) from common bean (Phaseolus vulgaris). Eight different classes of RGAs were obtained from nucleotide binding site (NBS)-based primers and seven from not previously described Toll/Interleukin-1 receptor-like (TIR)-based primers. Putative amino acid sequences of RGAs were significantly similar to R genes and contained additional conserved motifs. The NBS-type RGAs were classified in two subgroups according to the expected final residue in the kinase-2 motif. Eleven RGAs were mapped at 19 loci on eight linkage groups of the common bean genetic map constructed at Centro Internacional de Agricultura Tropical. Genetic linkage was shown for eight RGAs with partial resistance to anthracnose, angular leaf spot (ALS) and Bean golden yellow mosaic virus (BGYMV). RGA1 and RGA2 were associated with resistance loci to anthracnose and BGYMV and were part of two clusters of R genes previously described. A new major cluster was detected by RGA7 and explained up to 63.9% of resistance to ALS and has a putative contribution to anthracnose resistance. These results show the usefulness of RGAs as candidate genes to detect and eventually isolate numerous R genes in common bean.