Expression of EPL1 from Trichoderma atroviride in Arabidopsis Confers Resistance to Bacterial and Fungal Pathogens.

During plant interaction with beneficial microorganisms, fungi secrete a battery of elicitors that trigger plant defenses against pathogenic microorganisms. Among the elicitor molecules secreted by Trichoderma are cerato-platanin proteins, such as EPL1, from Trichoderma atroviride. In this study, Arabidopsis thaliana plants that express the TaEPL1 gene were challenged with phytopathogens to evaluate whether expression of EPL1 confers increased resistance to the bacterial pathogen Pseudomonas syringae and the necrotrophic fungus Botrytis cinerea. Infection assays showed that Arabidopsis EPL1-2, EPL1-3, EPL1-4 expressing lines were more resistant to both pathogens in comparison to WT plants. After Pseudomonas syringae infection, there were reduced disease symptoms (e.g., small chlorotic spots) and low bacterial titers in the three 35S::TaEPL1 expression lines. Similarly; 35S::TaEPL1 expression lines were more resistant to Botrytis cinerea infection, showing smaller lesion size in comparison to WT. Interestingly, an increase in ROS levels was detected in 35S::TaEPL1 expression lines when compared to WT. A higher expression of SA- and JA-response genes occurred in the 35S::TaEPL1 lines, which could explain the resistance of these EPL1 expression lines to both pathogens. We propose that EPL1 is an excellent elicitor, which can be used to generate crops with improved resistance to broad-spectrum diseases.

The entomopathogenic fungus Metarhizium anisopliae enhances Arabidopsis, tomato, and maize plant growth.

Species of the entomopathogenic fungi Metarhizium are used worldwide as biocontrol agents. Recently, other lifestyles have been associated with some Metarhizium species, which include their role as saprophytes, endophytes, and plant growth promoters. Herein, the effect of three Metarhizium anisopliae strains on the growth of Arabidopsis thaliana plantlets was evaluated using an in vitro split system. Arabidopsis fresh weight and total chlorophyll content significantly increased 7 days post-inoculation with the three Metarhizium anisopliae strains evaluated. The primary root length was promoted by all fungal strains without physical contact, whereas in direct contact primary root growth was inhibited. Volatile organic compounds identification revealed that during the interaction of Arabidopsis with Ma-20 and Ma-25 strains only ß-caryophyllene was produced, whereas in the Arabidopsis-Ma-28 interaction o-cymene was mainly emitted. The plant growth promoting effect induced by Metarhizium anisopliae strains was also achieved in Arabidopsis, tomato and maize plants grown in soil pots. Our results showed that three Metarhizium anisopliae strains were able to increase plant fresh weight, opening promising perspectives for field production, with the advantages of insect biocontrol and plant growth promotion induced by this species of fungus.

An interactome analysis reveals that Arabidopsis thaliana GRDP2 interacts with proteins involved in post-transcriptional processes.

The Arabidopsis thaliana glycine-rich domain protein 2 (AtGRDP2) gene encodes a protein of unknown function that is involved in plant growth and salt stress tolerance. The AtGRDP2 protein (787 aa, At4g37900) is constituted by three domains: a DUF1399 located at the N-terminus, a potential RNA Recognition Motif (RRM) in the central region, and a short glycine-rich domain at the C-terminus. Herein, we analyzed the subcellular localization of AtGRDP2 protein as a GFP translational fusion and found it was localized in the cytosol and the nucleus of tobacco leaf cells. Truncated versions of AtGRDP2 showed that the DUF1399 or the RRM domains were sufficient for nuclear localization. In addition, we performed a yeast two-hybrid split-ubiquitin assay (Y2H) to identify potential interactors for AtGRDP2 protein. The Y2H assay identified proteins associated with RNA binding functions such as PABN3 (At5g65260), EF-1α (At1g07920), and CL15 (At3g25920). Heterodimeric associations in planta between AtGRDP2 and its interactors were carried out by Bimolecular Fluorescence Complementation (BiFC) assays. The data revealed heterodimeric interactions between AtGRDP2 and PABN3 in the nucleus and AtGRDP2 with EF-1α in the cytosol, while AtGRDP2-CL15 associations occurred only in the chloroplasts. Finally, functional characterization of the protein-protein interaction regions revealed that both DUF1399 and RRM domains were key for heterodimerization with its interactors. The AtGRDP2 interaction with these proteins in different compartments suggests that this glycine-rich domain protein is involved in post-transcriptional processes.

Arabidopsis adc-silenced line exhibits differential defense responses to Botrytis cinerea and Pseudomonas syringae infection.

During plant-microbe interactions, polyamines participate in the plant defense response. Previously, we reported that silencing of ADC genes in Arabidopsis thaliana causes a drastic reduction of polyamine levels as well as increments in reactive oxygen species content. In this study, we examined the response of the adc-silenced line to Botrytis cinerea and Pseudomonas syringae infection. The adc-silenced line was more susceptible to Botrytis cinerea, showing larger lesion length and a higher incidence of fungal infection. Pre-treatments with putrescine reestablished the response of the adc-silenced line to Botrytis cinerea, resulting in a similar phenotype to the parental plant. Expression levels of defense-related genes were analyzed during fungal infection showing that the salicylic acid-induced gene PR1 was up-regulated, while the jasmonic acid-related genes LOX3 and PDF1.2, as well as, the camalexin biosynthetic gene PAD3 were down-regulated in the adc-silenced line. Furthermore, methyl jasmonate pre-treatments reduced Botrytis cinerea infection in the adc-silenced line. On the other hand, the adc-silenced line showed an increased resistance to Pseudomonas syringae infection. SA-related genes such as PR1, ZAT1.2, WRKY54 and WRKY70 were highly expressed in the adc-silenced line upon bacterial interaction. Our data show that the adc-silenced line has altered the defense-response against Botrytis cinerea and Pseudomonas syringae, that is consistent with deregulation of SA- and JA-mediated response pathways.

The cysteine-rich receptor-like protein kinase CRK28 modulates Arabidopsis growth and development and influences abscisic acid responses.

MAIN CONCLUSION: CRK28, a cysteine-rich receptor-like kinase, plays a role in root organogenesis and overall growth of plants and antagonizes abscisic acid response in seed germination and primary root growth. Receptor-like kinases (RLK) orchestrate development and adaptation to environmental changes in plants. One of the largest RLK groups comprises cysteine-rich receptor-like kinases (CRKs), for which the function of most members remains unknown. In this report, we show that the loss of function of CRK28 led to the formation of roots that are longer and more branched than the parental (Col-0) plantlets, and this correlates with an enhanced domain of the mitotic reporter CycB1:uidA in primary root meristems, whereas CRK28 overexpressing lines had the opposite phenotype, including slow root growth and reduced lateral root formation. Epidermal cell analyses revealed that crk28 mutants had reduced root hair length and increased trichome number, whereas 35S::CRK28 lines present primary roots with longer root hairs but lesser trichomes in leaves. The overall growth in soil of crk28 mutant and CRK28 overexpressing lines was reduced or enhanced, respectively, when compared to the parental (Col-0) seedlings, while germination, root growth and expression analyses of ABI3 and ABI5 further showed that CRK28 modulates ABA responses, which may be important to fine-tune plant morphogenesis. Our study unravels the participation of RLK signaling in root growth and epidermal cell differentiation.

The Arabidopsis-Trichoderma interaction reveals that the fungal growth medium is an important factor in plant growth induction.

Trichoderma spp colonizes the plant rhizosphere and provides pathogen resistance, abiotic stress tolerance, and enhance growth and development. We evaluated the Arabidopsis-Trichoderma interaction using a split system in which Trichoderma atroviride and Trichoderma virens were grown on PDA or MS medium. Arabidopsis growth was significantly increased at 3 and 5 days post-inoculation with both Trichoderma species, when the fungal strains were grown on PDA in split interaction. The analysis of DR5:uidA reporter line revealed a greater auxin accumulation in root tips when the fungi were grown on PDA in a split interaction. The root hair-defective phenotype of Arabidopsis rhd6 mutant was reverted with both Trichoderma species, even in split interactions. At 12 °C, Trichoderma species in split interactions were able to mitigate the effects of cold stress on the plant, and also Trichoderma induced the AtERD14 expression, a cold related gene. Volatile organic compounds analysis revealed that Trichoderma strains produce mainly sesquiterpenes, and that the type and abundance of these compounds was dependent on the fungal strain and the culture medium. Our results show that fungal nutrition is an important factor in plant growth in a split interaction.

Arabidopsis Polyamine oxidase-2 uORF is required for downstream translational regulation.

In eukaryotic mRNAs, small upstream open reading frames (uORFs) located in the 5'-untranslated region control the translation of the downstream main ORF. Polyamine oxidase (PAO) enzymes catalyze the oxidation of higher polyamines such as spermidine and spermine, and therefore contribute to the maintenance of intracellular polyamine content and to the regulation of physiological processes through their catabolic products. Recently, we reported that the Arabidopsis thaliana Polyamine Oxidase 2 (AtPAO2) is post-transcriptionally regulated by its 5'-UTR region through an uORF. In the present study, we analyzed whether the translation of the uORF is needed for the translational repression of the main ORF, and whether the inactivation of the uORF had an effect on the translational control mediated by polyamines. To this aim, we generated diverse single mutations in the uORF sequence; these mutant 5'-UTRs were fused to the GUS reporter gene, and tested in onion monolayer cells and A. thaliana transgenic seedlings. Removal of the start codon or introduction of a premature stop codon in the AtPAO2 uORF sequence abolished the negative regulation of the GUS expression exerted by the wild-type AtPAO2 uORF. An artificial uORF (32 amino acids in length) generated by the addition of a single nucleotide in AtPAO2 uORF proved to be less repressive than the wild-type uORF. Thus, our findings suggest that translation of the AtPAO2 uORF is necessary for the translational repression of the main ORF.

Further evidence from the effect of fungi on breaking Opuntia seed dormancy.

Recently, we found that fungi are involved in breaking seed dormancy of Opuntia streptacantha, and that the effect of fungi on seeds is species-specific. However, the effect of fungi on seed germination from other Opuntia spp has not been evaluated. Thus, we evaluated the effect of four fungal species (Penicillium chrysogenum, Phoma sp., Trichoderma harzianum, Trichoderma koningii) on the germination of Opuntia leucotricha, an abundant species in the Chihuahuan Desert, Mexico. We found that seeds inoculated with the four fungal species had higher germination than control seeds. Trichoderma spp. were the most effective. Our results strongly indicate that fungi are involved in breaking seed dormancy of O. leucotricha. Thus, we suggest that these fungi could promote seed germination from other Opuntia species.