Development of a droplet digital polymerase chain reaction (ddPCR) assay for the detection of Tomato brown rugose fruit virus (ToBRFV) in tomato and pepper seeds.

In the present study, a droplet digital PCR assay was developed for detection of Tomato brown rugose fruit virus, a new Tobamovirus of tomato and other solanaceous plants, which expands the diagnostic strategies for this pathogen. Candidate reference DNA material was also obtained to be employed as positive control in tomato and pepper samples. Recombinant plasmids encode for ToBRFV coat protein (CP-ToBRFV) gene and Solanum lycopersicum GAPDH fragments, and CP-ToBRFV and Capsicum annuum GAPDH. To our knowledge, this is the first report of ToBRFV detection in tomato and pepper seeds using ddPCR.

Genetic Diversity of Xylella fastidiosa in Mexican Vineyards.

Xylella fastidiosa is a xylem-inhabiting phytopathogenic bacterium that affects diverse agriculturally relevant crops. In Mexico, X. fastidiosa has been reported in the states of Baja California, Coahuila, and Querétaro. In order to determine the genetic diversity of this bacterium in Mexico, 408 grapevine samples were collected from the main producing states in México. For X. fastidiosa identification, real-time PCR and three-loci end-point PCR were employed. The genotyping of the subspecies was carried out using multilocus sequence typing and analysis, based on seven housekeeping genes: leuA, petC, malF, cysG, holC, nuoL, and gltT. The resulting sequences were compared with those present in extant databases. The presence of X. fastidiosa subsp. fastidiosa in the states of Baja California (sequence type 1), Coahuila (sequence type 1), and Querétaro was confirmed. The isolates from northern Mexico bear high similarity to grapevine isolates from the United States. However, the isolates from Querétaro showed significant differences with currently known sequences, showing that there is genetic variability among the X. fastidiosa subsp. fastidiosa populations from grapevines in northern and central Mexico.

Plant drought tolerance provided through genome editing of the trehalase gene.

Drought is one of the main abiotic factors that affect agricultural productivity, jeopardizing food security. Modern biotechnology is a useful tool for the generation of stress-tolerant crops, but its release and field-testing involves complex regulatory frameworks. However, gene editing technology mediated by the CRISPR/Cas9 system is a suitable strategy for plant breeding, which can lead to precise and specific modifications in the plant genome. The aim of the present work is to produce drought-tolerant plant varieties by modifying the trehalase gene. Furthermore, a new vector platform was developed to edit monocot and dicot genomes, by modifying vectors adding a streptomycin resistance marker for use with the hypervirulent Agrobacterium tumefaciens AGL1 strain. The gRNA design was based on the trehalase sequence in several species of the genus Selaginella that show drought tolerance. Arabidopsis thaliana carrying editions in the trehalase substrate-binding domain showed a higher tolerance to drought stress. In addition, a transient transformation system for gene editing in maize leaves was characterized.

Localized expression of antimicrobial proteins mitigates huanglongbing symptoms in Mexican lime.

Citrus huanglongbing (HLB) is a devastating disease associated with Candidatus Liberibacter asiaticus spp. (CLas), a bacterium restricted to the sieve tube system of the phloem that is transmitted by the psyllid vector, Diaphorina citri. In this study, the human antimicrobial peptides, lysozyme and ß-defensin 2, were targeted to the vascular tissue of Mexican lime (Citrus x aurantifolia [Christm.] Swingle) by fusion to a phloem-restricted protein. Localized expression was achieved, via Agrobacterium tumefaciens-mediated transformation of the stem, which led to protein expression and mobilization within the vascular tissue of heterotrophic tissues. HLB-infected plants were monitored for 360 days. Lower bacteria titers were observed in plants expressing either ß-defensin 2, lysozyme, or the combination thereof, and these plants had increased photosynthesis, compared to untreated control trees. Thus, targeting of antimicrobial proteins to the vascular tissue was effective in decreasing CLas titer, and alleviating citrus greening symptoms. Based on these findings, this strategy could be used to effectively treat plants that are already infected with bacterial pathogens that reside in the phloem translocation stream.

AtTCTP2, an Arabidopsis thaliana homolog of Translationally Controlled Tumor Protein, enhances in vitro plant regeneration.

The Translationally Controlled Tumor Protein (TCTP) is a central regulator of cell proliferation and differentiation in animals, and probably also in plants. Arabidopsis harbors two TCTP genes, AtTCTP1 (At3g16640), which is an important mitotic regulator, and AtTCTP2 (At3g05540), which is considered a pseudogene. Nevertheless, we have obtained evidence suggesting that this gene is functional. Indeed, a T-DNA insertion mutant, SALK_045146, displays a lethal phenotype during early rosette stage. Also, both the AtTCTP2 promoter and structural gene are functional, and heterozygous plants show delayed development. AtTCTP1 cannot compensate for the loss of AtTCTP2, since the accumulation levels of the AtTCTP1 transcript are even higher in heterozygous plants than in wild-type plants. Leaf explants transformed with Agrobacterium rhizogenes harboring AtTCTP2, but not AtTCTP1, led to whole plant regeneration with a high frequency. Insertion of a sequence present in AtTCTP1 but absent in AtTCTP2 demonstrates that it suppresses the capacity for plant regeneration; also, this phenomenon is enhanced by the presence of TCTP (AtTCTP1 or 2) in the nuclei of root cells. This confirms that AtTCTP2 is not a pseudogene and suggests the involvement of certain TCTP isoforms in vegetative reproduction in some plant species.

A 2-component system is involved in the early stages of the Pisolithus tinctorius-Pinus greggii symbiosis.

Ectomycorrhizal symbiosis results in profound morphological and physiological modifications in both plant and fungus. This in turn is the product of differential gene expression in both co-symbionts, giving rise to specialized cell types capable of performing novel functions. During the precolonization stage, chemical signals from root exudates are sensed by the ectomycorrizal fungus, and vice versa, which are in principle responsible for the observed change in the developmental symbionts program. Little is known about the molecular mechanisms involved in the signaling and recognition between ectomycorrhizal fungi and their host plants. In the present work, we characterized a novel lactone, termed pinelactone, and identified a gene encoding for a histidine kinase in Pisolithus tictorius, which function is proposed to be the perception of the aforementioned metabolites. In this study, the use of closantel, a specific inhibitor of histidine kinase phosphorylation, affected the capacity for fungal colonization in the symbiosis between Pisolithus tinctorius and Pinus greggii, indicating that a 2-component system (TCS) may operate in the early events of plant-fungus interaction. Indeed, the metabolites induced the accumulation of Pisolithus tinctorius mRNA for a putative histidine kinase (termed Pthik1). Of note, Pthik1 was able to partially complement a S. cerevisiae histidine kinase mutant, demonstrating its role in the response to the presence of the aforementioned metabolites. Our results indicate a role of a 2-component pathway in the early stages of ectomycorrhizal symbiosis before colonization. Furthermore, a novel lactone from Pinus greggii root exudates may activate a signal transduction pathway that contributes to the establishment of the ectomycorrhizal symbiosis.

Phylogenetic and structural analysis of translationally controlled tumor proteins.

The translationally controlled tumor protein (TCTP) is conserved in all eukaryotes studied thus far. Recent evidence points to an important role for TCTP in the induction of cell proliferation in animals through an interaction with G proteins. TCTP may also constitute an intercellular secreted signal that modulates the immune response in the vertebrates. Because of its sequence conservation and ubiquity, the analysis of its amino acid sequence divergence between different taxa may provide insight into the structural constraints on the evolution of this protein. In the present study, we analyzed the phylogeny of TCTP sequences from a wide range of organisms and found that, with some exceptions, the groupings formed were consistent with the evolutionary history. Indeed, at the level of lower-order taxa, the groupings are in agreement with their established phylogeny, thus indicating that the substitution rates of the TCTP residues varied evenly between members of the same clade. Predicted three-dimensional structures of representative TCTPs, based on the reported 3D structure of Schizosaccharomyces pombe, indicated that these proteins are highly conserved among diverse taxonomic groups. However, analysis of the primary structure indicated subtle differences in the domain-forming pocket that potentially interacts with G proteins, particularly among Diplomonadidae, Apicomplexa, and other parasites of vertebrates. These differences support the notion that these specific TCTPs could block the normal immune response by acting as dominant negative mutants. Structural differences were also observed in a reported sequence of TCTP from Plasmodium knowlesi, in which the presence of an extra alpha-helix could also interfere in the interaction with G proteins.