Bacillus Strains as Effective Biocontrol Agents Against Phytopathogenic Bacteria and Promoters of Plant Growth.

Modern crop production relies on the application of chemical pesticides and fertilizers causing environmental and economic challenges. In response, less environmentally impactful alternatives have emerged such as the use of beneficial microorganisms. These microorganisms, particularly plant growth-promoting bacteria (PGPB), have demonstrated their ability to enhance plant growth, protect against various stresses, and reduce the need for chemical inputs. Among the PGPB, Bacillus species have garnered attention due to their adaptability and commercial potential. Recent reports have highlighted Bacillus strains as biocontrol agents against phytopathogenic bacteria while concurrently promoting plant growth. We also examined Bacillus plant growth-promoting abilities in Arabidopsis thaliana seedlings. In this study, we assessed the potential of various Bacillus strains to control diverse phytopathogenic bacteria and inhibit quorum sensing using Chromobacterium violaceum as a model system. In conclusion, our results suggest that bacteria of the genus Bacillus hold significant potential for biotechnological applications. This includes developments aimed at reducing agrochemical use, promoting sustainable agriculture, and enhancing crop yield and protection.

Evolutionary and Structural Analysis of PP16 in Viridiplantae.

Members of the phloem protein 16 (PP16) gene family are induced by elicitors in rice and the corresponding proteins from cucurbits, which display RNA binding and intercellular transport activities, are accumulated in phloem sap. These proteins facilitate the movement of protein complexes through the phloem translocation flow and may be involved in the response to water deficit, among other functions. However, there is scant information regarding their function in other plants, including the identification of paralog genes in non-vascular plants and chlorophytes. In the present work, an evolutionary and structural analysis of the PP16 family in green plants (Viridiplantae) was carried out. Data mining in different databases indicated that PP16 likely originated from a larger gene present in an ancestral lineage that gave rise to chlorophytes and multicellular plants. This gene encodes a protein related to synaptotagmin, which is involved in vesicular transport in animal systems, although other members of this family play a role in lipid turnover in endomembranes and organelles. These proteins contain a membrane-binding C2 domain shared with PP16 proteins in vascular plants. In silico analysis of the predicted structure of the PP16 protein family identified several ß-sheets, one α-helix, and intrinsically disordered regions. PP16 may have been originally involved in vesicular trafficking and/or membrane maintenance but specialized in long-distance signaling during the emergence of the plant vascular system.

Transcriptomic Analysis of the Host Response to Mild and Severe CTV Strains in Naturally Infected Citrus sinensis Orchards.

Citrus tristeza virus (CTV) is an important threat to the global citrus industry, causing severe economic losses worldwide. The disease management strategies are focused on vector control, tree culling, and the use of resistant varieties and rootstocks. Sweet orange (Citrus sinensis) trees showing either severe or mild CTV symptoms have been observed in orchards in Veracruz, Mexico, and were probably caused by different virus strains. To understand these symptomatic differences, transcriptomic analyses were conducted using asymptomatic trees. CTV was confirmed to be associated with infected plants, and mild and severe strains were successfully identified by a polymorphism in the coat protein (CP) encoding gene. RNA-Seq analysis revealed more than 900 significantly differentially expressed genes in response to mild and severe strains, with some overlapping genes. Importantly, multiple sequence reads corresponding to Citrus exocortis viroid and Hop stunt viroid were found in severe symptomatic and asymptomatic trees, but not in plants with mild symptoms. The differential gene expression profiling obtained in this work provides an overview of molecular behavior in naturally CTV-infected trees. This work may contribute to our understanding of citrus-virus interaction in more natural settings, which can help develop strategies for integrated crop management.

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.

An Ancestry Perspective of the Evolution of PBS1 Proteins in Plants.

The AVRPPHB SUSCEPTIBLE1 (PBS1) and RESISTANCE TO PSEUDOMONAS SYRINGAE 5 (RPS5) proteins are involved in signal transduction to evoke innate plant immune response. In Arabidopsis, PBS1 is cleaved by the AvrPphB (Pseudomonas phaseolicola Avirulence protein B) protease, activating RPS5 and turning in a hypersensitive response (HR). We searched for PBS1 orthologs to trace their origin and evolution. PBS1 orthologs were found in embryophytes and in other plant taxa but with lower similarity. PBS1 phylogenetic analysis indicates high divergence, suggesting that the decoy function described for Arabidopsis PBS1 might be associated with a small fraction of orthologs. Ancestral reconstruction analysis suggests an elevated diversity in the amino acid sequence within the described motifs. All the orthologs contain the conserved PBS1 kinase subdomains, whereas the cleavage motif is present in several embryophyte orthologs but absent in most other taxa. The putative resistance recognition motifs in PBS1 orthologs are highly diverse. PBS1 cleavage site motif is exposed in some 3D structure predictions, whereas it is not in others, suggesting different modes of regulation and functions in PBS1 orthologs. Our findings suggest that PBS1 originated in the lineage that gave rise to embryophytes, with the angiosperm sequences forming a separate clade from pteridophyte proteins.

A novel intracellular nitrogen-fixing symbiosis made by Ustilago maydis and Bacillus spp.

We observed that the maize pathogenic fungus Ustilago maydis grew in nitrogen (N)-free media at a rate similar to that observed in media containing ammonium nitrate, suggesting that it was able to fix atmospheric N2 . Because only prokaryotic organisms have the capacity to reduce N2 , we entertained the possibility that U. maydis was associated with an intracellular bacterium. The presence of nitrogenase in the fungus was analyzed by acetylene reduction, and capacity to fix N2 by use of (15) N2 . Presence of an intracellular N2 -fixing bacterium was analyzed by PCR amplification of bacterial 16S rRNA and nifH genes, and by microscopic observations. Nitrogenase activity and (15) N incorporation into the cells proved that U. maydis fixed N2 . Light and electron microscopy, and fluorescence in situ hybridization (FISH) experiments revealed the presence of intracellular bacteria related to Bacillus pumilus, as evidenced by sequencing of the PCR-amplified fragments. These observations reveal for the first time the existence of an endosymbiotic N2 -fixing association involving a fungus and a bacterium.

Resistance to stem rust Ug99 in six bread wheat cultivars maps to chromosome 6DS.

KEY MESSAGE: Identified SSR markers ( Xcfd49 and Xbarc183 ) linked with stem rust resistance for efficient use in marker-assisted selection and stacking of resistance genes in wheat breeding programs. More than 80 % of the worldwide wheat (Triticum aestivum L.) area is currently sown with varieties susceptible to the Ug99 race group of stem rust fungus. However, wheat lines Niini, Tinkio, Coni, Pfunye, Blouk, and Ripper have demonstrated Ug99 resistance at the seedling and adult plant stages. We mapped stem rust resistance in populations derived from crosses of a susceptible parent with each of the resistant lines. The segregation of resistance in each population indicated the presence of a single gene. The resistance gene in Niini mapped to short arm of chromosome 6D and was flanked by SSR markers Xcfd49 at distances of 3.9 cM proximal and Xbarc183 8.4 cM distal, respectively. The chromosome location of this resistance was validated in three other populations: PBW343/Coni, PBW343/Tinkio, and Cacuke/Pfunye. Resistance initially postulated to be conferred by the SrTmp gene in Blouk and Ripper was also linked to Xcfd49 and Xbarc183 on 6DS, but it was mapped proximal to Xbarc183 at a similar position to previously mapped genes Sr42 and SrCad. Based on the variation in diagnostic marker alleles, it is possible that Niini and Pfunye may carry different resistance genes/alleles. Further studies are needed to determine the allelic relationships between various genes located on chromosome arm 6DS. Our results provide valuable molecular marker and genetic information for developing Ug99 resistant wheat varieties in diverse germplasm and using these markers to tag the resistance genes in wheat breeding.

Long-Distance Movement of Solanum tuberosum Translationally Controlled Tumor Protein (StTCTP) mRNA.

Long-distance signaling molecules in plants, including different RNA species, play a crucial role in the development and environmental responses. Among these mobile signals, the Translationally Controlled Tumor Protein (TCTP) mRNA is one of the most abundant. TCTP regulates cell-cycle progression and programmed cell death and is involved in responses to abiotic and biotic stress as well as plant regeneration, among other functions. Considering that the ability to induce plant regeneration is linked to a possible role of TCTP in vegetative propagation and asexual reproduction, we analyzed TCTP overexpression in a solanaceous plant model that can reproduce asexually by regeneration from stolons and tubers. Therefore, in this study, the effect of transient expression of Solanum tuberosum TCTP (StTCTP) on tuber development and vegetative propagation was described. StTCTP mRNA was shown to be transported long-distance. Additionally, transient overexpression of StTCTP resulted in sprouts with a greater diameter compared to control plants. Furthermore, the early stages of tuberization were induced compared to control plants, in which only mature tubers were observed. These results suggest a role of TCTP in vegetative propagation and asexual reproduction.

The Characterization of Melanaphis sacchari Microbiota and Antibiotic Treatment Effect on Insects.

Insects are under constant selective pressure, which has resulted in adaptations to novel niches such as crops. This is the case of the pest Melanaphis sacchari, the sugarcane aphid, native to Africa and currently spreading worldwide. The aphid undergoes successful parthenogenesis, causing important damage to a variety of crops and leading to important economic losses for farmers. A natural M. sacchari population grown in sorghum was studied to identify its microbiome through the sequencing of its 16S rDNA metagenome. A high proportion of Proteobacteria, followed by Firmicutes, Bacteroidetes, and Actinobacteria, was observed. We also detected Wolbachia, which correlates with the asexual reproduction of its host. M. sacchari was challenged in a bioassay with the antibiotics oxytetracycline and streptomycin, resulting in a dose-dependent decay of its survival rate. The possibility of controlling this pest by altering its microbiota is proposed.

Starting from scratch: Step-by-step development of diagnostic tests for SARS-CoV-2 detection by RT-LAMP.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. Public health strategies to reduce viral transmission are based on widespread diagnostic testing to detect and isolate contagious patients. Several reverse transcription (RT)-PCR tests, along with other SARS-CoV-2 diagnostic assays, are available to attempt to cover the global demand. Loop-mediated isothermal amplification (LAMP) based methods have been established as rapid, accurate, point of care diagnostic tests for viral infections; hence, they represent an excellent alternative for SARS-CoV-2 detection. The aim of this study was to develop and describe molecular detection systems for SARS-CoV-2 based on RT-LAMP. Recombinant DNA polymerase from Bacillus stearothermophilus and thermostable engineered reverse transcriptase from Moloney Murine Leukemia Virus were expressed using a prokaryotic system and purified by fast protein liquid chromatography. These enzymes were used to set up fluorometric real time and colorimetric end-point RT-LAMP assays. Several reaction conditions were optimized such as reaction temperature, Tris-HCl concentration, and pH of the diagnostic tests. The key enzymes for RT-LAMP were purified and their enzymatic activity was determined. Standardized reaction conditions for both RT-LAMP assays were 65°C and a Tris-HCl-free buffer at pH 8.8. Colorimetric end-point RT-LAMP assay was successfully used for viral detection from clinical saliva samples with 100% sensitivity and 100% specificity compared to the results obtained by RT-qPCR based diagnostic protocols with Ct values until 30. The developed RT-LAMP diagnostic tests based on purified recombinant enzymes allowed a sensitive and specific detection of the nucleocapsid gene of SARS-CoV-2.

Vascular expression in Arabidopsis is predicted by the frequency of CT/GA-rich repeats in gene promoters.

Phloem-transported signals play an important role in regulating plant development and in orchestrating responses to environmental stimuli. Among such signals, phloem-mobile RNAs have been shown to play an important role as long-distance signaling agents. At maturity, angiosperm sieve elements are enucleate, and thus transcripts in the phloem translocation stream probably originate from the nucleate companion cells. In the present study, a pumpkin (Cucurbita maxima) phloem transcriptome was used to test for the presence of common motifs within the promoters of this unique set of genes, which may function to coordinate expression in cells of the vascular system. A bioinformatics analysis of the upstream sequences from 150 Arabidopsis genes homologous to members of the pumpkin phloem transcriptome identified degenerate sequences containing CT/GA- and GT/CA-rich motifs that were common to many of these promoters. Parallel studies performed on genes shown previously to be expressed in phloem tissues identified similar motifs. An expanded analysis, based on homologs of the pumpkin phloem transcriptome from cucumber (Cucumis sativus), identified similar sets of common motifs within the promoters of these genes. Promoter analysis offered support for the hypothesis that these motifs regulate expression within the vascular system. Our findings are discussed in terms of a role for these motifs in coordinating gene expression within the companion cell/sieve element system. These motifs could provide a useful bioinformatics tool for genome-wide screens on plants for which phloem tissues cannot readily be obtained.

The NAC Transcription Factor ANAC087 Induces Aerial Rosette Development and Leaf Senescence in Arabidopsis.

CmNACP1 mRNA has been shown to move long distance through the phloem in Cucurbita maxima (pumpkin) and through a graft junction. Whereas the phloem transport of several different mRNAs has been documented in other systems as well, its function remains, for most of these RNAs, largely unknown. To gain insight into the possible role of these RNAs, we searched for the closest homologs of CmNACP1 in Arabidopsis, a model plant much more amenable for analysis. A phylogenetic approach using the predicted NAC domain indicated that ANAC059, ANAC092, ANAC079, ANAC100, ANAC046, and ANAC087 form a single clade with CmNACP1. In the present work, we analyzed the possible function of the ANAC087 gene in more detail. The promoter region of this gene directed expression in the vasculature, and also in trichomes, stem, apexes, and developing flowers which supports the notion that ANAC087 and CmNACP1 are orthologs. Overexpression of the ANAC087 gene induced increased branching in inflorescence stem, and also development of ectopic or aerial rosettes in T1 and T2 plants. Furthermore, overexpression of ANAC087 leads to accelerated leaf senescence in 44 days post-germination (dpg). Interestingly, a similar phenotype was observed in plants expressing the ANAC087 gene upstream region, also showing an increase in ANAC087 transcript levels. Finally, the results shown in this work indicate a role for ANAC087 in leaf senescence and also in rosette development.

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.

Engineering Macromolecular Trafficking Into the Citrus Vasculature.

The plant vasculature is a central organ for long-distance transport of nutrients and signaling molecules that coordinate vegetative and reproductive processes, and adaptation response mechanisms to biotic and abiotic stress. In angiosperms, the sieve elements are devoid of nuclei, thus depending on the companion cells for the synthesis of RNA and proteins, which constitute some of the systemic signals that coordinate these processes. Massive analysis approaches have identified proteins and RNAs that could function as long-range signals in the phloem translocation stream. The selective translocation of such molecules could occur as ribonucleoprotein complexes. A key molecule facilitating this movement in Cucurbitaceae is the phloem protein CmPP16, which can facilitate the movement of RNA and other proteins into the sieve tube. The CmPP16 ortholog in Citrus CsPP16 was characterized in silico to determine its potential capacity to associate with other mobile proteins and its enrichment in the vascular tissue. The systemic nature of CsPP16 was approached by evaluating its capacity to provide phloem-mobile properties to antimicrobial peptides (AMPs), important in the innate immune defense. The engineering of macromolecular trafficking in the vasculature demonstrated the capacity to mobilize translationally fused peptides into the phloem stream for long-distance transport. The translocation into the phloem of AMPs could mitigate the growth of Candidatus Liberibacter asiaticus, with important implications for crop defense; this system also opens the possibility of translocating other molecules to modulate traits, such as plant growth, defense, and plant productivity.

Bioinformatic-based approach for mutagenesis of plant immune Tm-22 receptor to confer resistance against tomato brown rugose fruit virus (ToBRFV).

Nucleotide-binding leucine-rich repeat (NLR) plant immune receptors mediate the recognition and activation of defense signaling pathways in response to intra- and extracellular pathogens. Several NLR such as Tm-2 and Tm-22 have been introgressed into commercial solanaceous varieties to confer protection against different tobamoviruses. Particularly, Tm-22 was used during recent decades to confer resistance against tobacco mosaic virus, tomato mottle mosaic virus and tomato mosaic virus, which recognizes the viral movement protein (MP). However, tomato brown rugose fruit virus(ToBRFV), a novel tobamovirus, can avoid the protection conferred by Tm-22 due to the presence of key substitutions in the MP. The aim of this work was to identify the key amino acid residues involved in the interaction between Tm-22 and ToBRFV MP through bioinformatic analyses, and to identify potential Tm-22 mutations that could generate greater binding affinity. In silico 3D structure prediction, molecular docking, and computational affinity methods were performed. We predicted that R350, H384 and K385 Tm-22 residues are relevant for the interaction with MP, and two mutations (H384W and K385L) were identified as putative sites to increase the affinity of Tm-22 to the MP with the potential elicitation of resistance against ToBRFV.

Combined Subcutaneous-Intranasal Immunization With Epitope-Based Antigens Elicits Binding and Neutralizing Antibody Responses in Serum and Mucosae Against PRRSV-2 and SARS-CoV-2.

New vaccine design approaches, platforms, and immunization strategies might foster antiviral mucosal effector and memory responses to reduce asymptomatic infection and transmission in vaccinated individuals. Here, we investigated a combined parenteral and mucosal immunization scheme to induce local and serum antibody responses, employing the epitope-based antigens 3BT and NG19m. These antigens target the important emerging and re-emerging viruses PRRSV-2 and SARS-CoV-2, respectively. We assessed two versions of the 3BT protein, which contains conserved epitopes from the GP5 envelope protein of PRRSV-2: soluble and expressed by the recombinant baculovirus BacDual-3BT. On the other hand, NG19m, comprising the receptor-binding motif of the S protein of SARS-CoV-2, was evaluated as a soluble recombinant protein only. Vietnamese mini-pigs were immunized employing different inoculation routes: subcutaneous, intranasal, or a combination of both (s.c.-i.n.). Animals produced antigen-binding and neut1ralizing antibodies in serum and mucosal fluids, with varying patterns of concentration and activity, depending on the antigen and the immunization schedule. Soluble 3BT was a potent immunogen to elicit binding and neutralizing antibodies in serum, nasal mucus, and vaginal swabs. The vectored immunogen BacDual-3BT induced binding antibodies in serum and mucosae, but PRRSV-2 neutralizing activity was found in nasal mucus exclusively when administered intranasally. NG19m promoted serum and mucosal binding antibodies, which showed differing neutralizing activity. Only serum samples from subcutaneously immunized animals inhibited RBD-ACE2 interaction, while mini-pigs inoculated intranasally or via the combined s.c.-i.n. scheme produced subtle neutralizing humoral responses in the upper and lower respiratory mucosae. Our results show that intranasal immunization, alone or combined with subcutaneous delivery of epitope-based antigens, generates local and systemic binding and neutralizing antibodies. Further investigation is needed to evaluate the capability of the induced responses to prevent infection and reduce transmission.

Geminivirus mixed infection on pepper plants: synergistic interaction between PHYVV and PepGMV.

BACKGROUND: PHYVV and PepGMV are plant viruses reported in Mexico and Southern US as causal agents of an important pepper disease known as "rizado amarillo". Mixed infections with PHYVV and PepGMV have been reported in several hosts over a wide geographic area. Previous work suggested that these viruses might interact at the replication and/or movement level in a complex manner. The aim of present report was to study some aspects of a synergistic interaction between PHYVV and PepGMV in pepper plants. These include analyses of symptom severity, viral DNA concentration and tissue localization of both viruses in single and mixed infections. RESULTS: Mixed infections with PepGMV and PHYVV induced symptoms more severe than those observed in single viral infections. Whereas plants infected with either virus (single infection) presented a remission stage with a corresponding decrease in viral DNA levels, double-infected plants did not present symptom remission and both viral DNA concentrations dramatically increased. In situ hybridization experiments revealed that both viruses are restricted to the vascular tissue. Interestingly, the amount of viral DNA detected was higher in plants inoculated with PepGMV than that observed in PHYVV-infected plants. During mixed infections, the location of both viruses remained similar to the one observed in single infections, although the number of infected cells increases. Infections with the tripartite mixture PHYVV (A+B) + PepGMV A produced a similar synergistic infection to the one observed after inoculation with both full viruses. On the contrary, tripartite mixture PepGMV (A+B) + PHYVV A did not produce a synergistic interaction. In an attempt to study the contribution of individual genes to the synergism, several mutants of PHYVV or PepGMV were inoculated in combination with the corresponding wild type, second virus (wt PepGMV or wt PHYVV). All combinations tested resulted in synergistic infections, with exception of the TrAP mutant of PepGMV (PepGMV TrAP-) + PHYVV. CONCLUSION: In this report, we have demonstrated that synergistic interaction between PHYVV and PepGMV during a mixed infection is mainly due to an increased DNA concentration of both viruses, without any noticeable effect on the localization of either virus on infected plant tissue. Our results have shown that the viral component A from PepGMV is important for synergism during PHYVV-PepGMV mixed infections.

Adsorption of Recombinant Human ß-Defensin 2 and Two Mutants on Mesoporous Silica Nanoparticles and Its Effect against Clavibacter michiganensis subsp. michiganensis.

Solanum lycopersicum L. is affected among other pests and diseases, by the actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm), causing important economic losses worldwide. Antimicrobial peptides (AMPs) are amphipathic cationic oligopeptides with which the development of pathogenic microorganisms has been inhibited. Therefore, in this study, we evaluate antimicrobial activity of mesoporous silica nanoparticles (MSN5.4) loaded with human ß-defensin-2 (hßD2) and two mutants (TRX-hßD2-M and hßD2-M) against Cmm. hßD2, TRX-hßD2-M and hßD2-M presented a half-maximum inhibitory concentration (IC50) of 3.64, 1.56 and 6.17 µg/mL, respectively. MSNs had average particle sizes of 140 nm (SEM) and a tunable pore diameter of 4.8 up to 5.4 nm (BJH). AMPs were adsorbed more than 99% into MSN and a first release after 24 h was observed. The MSN loaded with the AMPs inhibited the growth of Cmm in solid and liquid media. It was also determined that MSNs protect AMPs from enzymatic degradation when the MSN/AMPs complexes were exposed to a pepsin treatment. An improved AMP performance was registered when it was adsorbed in the mesoporous matrix. The present study could expand the applications of MSNs loaded with AMPs as a biological control and provide new tools for the management of phytopathogenic microorganisms.

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.

Wide distribution of the Ustilago maydis-bacterium endosymbiosis in naturally infected maize plants.

We have previously described that laboratory strains of Ustilago maydis, a fungal pathogen of maize and its ancestor teosinte, harbor an intracellular bacterium that enables the fungus to fix nitrogen. However, it is not clear whether other strains isolated from nature also harbor endosymbiotic bacteria, and whether these fix nitrogen for its host. In the present study, we isolated U. maydis strains from naturally infected maize. All the isolated strains harbored intracellular bacteria as determined by PCR amplification of the 16S rRNA gene, and some of them showed capacity to fix nitrogen. That these are truly bacterial endosymbionts were shown by the fact that, after thorough treatments with CuSO4 followed by serial incubations with antibiotics, the aforementioned bacterial gene was still amplified in treated fungi. In all, these data support the notion that U. maydis-bacterium endosymbiosis is a general phenomenon in this species.