Differential expression of pathogen-responsive genes encoding two types of glycine-rich proteins in barley

Gene-specific probes (3'' ends of cDNAs) were obtained from barley cDNAs encoding two types of glycine-rich proteins: HvGRP2, characterized by a cytokeratin-like and a cysteine-rich domain, and HvGRP3, whose main feature was an RNA-binding domain. Expression of genes Hvgrp2 and Hvgrp3, which are present at one (or two) copies per haploid genome, was ubiquitous and gene Hvgrp3 was under light/darkness modulation. Cold treatment increased Hvgrp2 and Hvgrp3 mRNA levels. Methyl jasmonate (10 M) switched off the two genes. Expression of Hvgrp2, but not that of Hvgrp3, was induced by ethylene treatment (100 ppm). Fungal pathogens Erysiphe graminis and Rhynchosporium secalis increased the mRNAs levels of the two genes, both in compatible and in incompatible interactions, while bacterial pathogens did no

YODA Kinase Controls a Novel Immune Pathway of Tomato Conferring Enhanced Disease Resistance to the Bacterium Pseudomonas syringae

© 2020 Téllez, Muñoz-Barrios, Sopeña-Torres, Martín-Forero, Ortega, Pérez, Sanz, Borja, de Marcos, Nicolas, Jahrmann, Mena, Jordá and Molina.Mitogen-activated protein kinases (MAPK) play pivotal roles in transducing developmental cues and environmental signals into cellular responses through pathways initiated by MAPK kinase kinases (MAP3K). AtYODA is a MAP3K of Arabidopsis thaliana that controls stomatal development and non-canonical immune responses. Arabidopsis plants overexpressing a constitutively active YODA protein (AtCA-YDA) show broad-spectrum disease resistance and constitutive expression of defensive genes. We tested YDA function in crops immunity by heterologously overexpressing AtCA-YDA in Solanum lycopersicum. We found that these tomato AtCA-YDA plants do not show developmental phenotypes and fitness alterations, except a reduction in stomatal index, as reported in Arabidopsis AtCA-YDA plants. Notably, AtCA-YDA tomato plants show enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and constitutive upregulation of defense-associated genes, corroborating the functionality of YDA in tomato immunity. This function was further supported by generating CRISPR/Cas9-edited tomato mutants impaired in the closest orthologs of AtYDA [Solyc08g081210 (SlYDA1) and Solyc03g025360 (SlYDA2)]. Slyda1 and Slyda2 mutants are highly susceptible to P. syringae pv. tomato DC3000 in comparison to wild-type plants but only Slyda2 shows altered stomatal index. These results indicate that tomato orthologs have specialized functions and support that YDA also regulates immune responses in tomato and may be a trait for breeding disease resistance.This work was supported by grants BIO2015-64077-R of the Spanish Ministry of Economy and Competitiveness (MINECO), RTI2018-096975-B-I00 of Spanish Ministry of Science, Innovation and Universities, and grant P190050072 of Plant Response Biotech SL to AMo. Research in the Montaña Mena’s laboratory is supported by the MINECO (PPII10-0194-4164) and the Junta de Comunidades de Castilla-La Mancha (SBPLY/17/180501/000394), complemented with EU FEDER funds. JT was financially supported by a PhD fellow of the FPI program from MINECO (BES-2016-076708). AM-F and AO were recipients of research and PhD fellowships from JCCM

Overexpression of a SDD1-Like Gene From Wild Tomato Decreases Stomatal Density and Enhances Dehydration Avoidance in Arabidopsis and Cultivated Tomato

Stomata are microscopic valves formed by two guard cells flanking a pore, which are located on the epidermis of most aerial plant organs and are used for water and gas exchange between the plant and the atmosphere. The number, size and distribution of stomata are set during development in response to changing environmental conditions, allowing plants to minimize the impact of a stressful environment. In Arabidopsis, STOMATAL DENSITY AND DISTRIBUTION 1 (AtSDD1) negatively regulates stomatal density and optimizes transpiration and water use efficiency (WUE). Despite this, little is known about the function of AtSDD1 orthologs in crop species and their wild stress-tolerant relatives. In this study, SDD1-like from the stress-tolerant wild tomato Solanum chilense (SchSDD1-like) was identified through its close sequence relationship with SDD1-like from Solanum lycopersicum and AtSDD1. Both Solanum SDD1-like transcripts accumulated in high levels in young leaves, suggesting that they play a role in early leaf development. Arabidopsis sdd1-3 plants transformed with SchSDD1-like under a constitutive promoter showed a significant reduction in stomatal leaf density compared with untransformed sdd1-3 plants. Additionally, a leaf dehydration shock test demonstrated that the reduction in stomatal abundance of transgenic plants was sufficient to slow down dehydration. Overexpression of SchSDD1-like in cultivated tomato plants decreased the stomatal index and density of the cotyledons and leaves, and resulted in higher dehydration avoidance. Taken together, these results indicate that SchSDD1-like functions in a similar manner to AtSDD1 and suggest that Arabidopsis and tomatoes share this component of the stomatal development pathway that impinges on water status

Hyperthermophilic aldolases as biocatalyst for C–C bond formation: rhamnulose 1-phosphate aldolase from Thermotoga maritima

The TM1072 gene from Thermotoga maritima codifies for a putative form of a rhamnulose-1-phosphate aldolase (Rha-1PATm). To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified enzyme was activated by Co2+ as a divalentmetal ion cofactor, instead of Zn2+ as its E. coli homologue, and exhibited a maximum of activity at 95 °C. Furthermore, the enzyme displayed a high stability against extreme reaction conditions, retaining 90 % of its activity in the presence of 40 % of acetonitrile and showing a half-life greater than 3 h at 115 °C. The kinetic parameters at room temperature (R/T) were also studied; the KM was calculated to be 3.6±0.33 mM, while kcat/KM was found to be 0.7× 103 s−1 M−1. Given these characteristics, Rha-1PA Tm is an attractive enzyme for use as a biocatalyst for industrial applications, offering intriguing possibilities for practical biocatalysis.We thank the Spanish Ministerio de Economía y Competitividad (Grant PI11/01436) and Comunidad de Madrid (Grant S2009/PPQ-1752) for financial support. I. O.-G. is a JAEPredoc fellow from CSIC.Peer reviewe

Catalytic promiscuity modulated by metal switching: evolution of new enzyme functions

1 página.-- Congreso organizado por la Sociedad Española de Biotecnología.Peer reviewe

An endosperm-specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin-box of a native B-hordein promoter in barley endosperm

A cDNA encoding a DNA‐binding protein of the DOF class of transcription factors was isolated from a barley endosperm library. The deduced amino acid sequence for the corresponding protein is 94% identical through the DOF domain to the prolamin‐box (P‐box) binding factor PBF from maize. The gene encoding the barley PBF (BPBF) maps to chromosome 7H, and its expression is restricted to the endosperm where it precedes that of the hordein genes. The BPBF expressed in bacteria as a GST‐fusion binds a P‐box 5′‐TGTAAAG‐3′ containing oligonucleotide derived from the promoter region of an Hor2 gene. Binding was prevented when the P‐box motif was mutated to 5′‐TGTAgAc‐3′. A P‐box binding activity, present in barley and wheat endosperm nuclei, interacted similarly to BPBF with this synthetic oligonucleotide, and the binding was abolished by 1,10‐phenanthroline. Transient expression experiments in developing barley endosperms demonstrate that BPBF transactivates transcription from the P‐box element of a native Hor2 promoter and that direct binding of BPBF to its target site is essential for transactivation since mutations in the DOF DNA‐binding domain or in the P‐box motif of this promoter abolished both binding and transactivation. Evidence was also obtained for the presence in wheat of a Pbf homologue having similar DNA‐binding properties to that of BPBF. These results strongly implicate this endosperm‐specific DOF protein from barley as an important activator of hordein gene expression and suggest the evolutionary conservation of the Pbf gene function among small grain cereals

Dynamic analysis of epidermal cell divisions identifies specific roles for COP10 in Arabidopsis stomatal lineage development

Stomatal development in Arabidopsis thaliana has been linked to photoreceptor-perceived light through several components of the photomorphogenic switch, whose lack of function is often seedling-lethal. CONSTITUTIVE PHOTOMORPHOGENIC 10 (COP10) is an important component of this switch, its loss of function producing stomatal clusters. Exploiting the reduced lethality of the cop10-1 mutant we characterized the developmental basis of its stomatal phenotype. Constitutive, light-independent stomata overproduction accounts for half of cop10-1 stomatal abundance and appears very early in development. Clusters are responsible for the remaining stomata excess and build-up progressively at later stages. Serial impressions of living cotyledon epidermis allowed a dynamic, quantitative analysis of stomatal lineage types by reconstructing their division histories. We found that COP10 adjusts the initiation frequency and extension of stomatal lineages (entry and amplifying asymmetric divisions) and represses stomatal fate in lineage cells; COP10 also supervises the orientation of spacing divisions in satellite lineages, preventing the appearance of stomata in contact. Aberrant accumulation of the proliferating stomatal lineage cell marker TMMpro::TMM-GFP showed that the abundant cop10-1 stomatal lineages maintained extended and ectopic competence for stomatal fate. Expression of stomatal development master genes suggests that the mutant does not bypass major molecular actors in this process. cop10-1 first leaf produces trichomes and apparently normal pavement cells, but functionally and morphologically aberrant stomata; COP10 operates genetically in parallel to the stomatal repressor SDD1 and does not generally affect epidermal cell differentiation, but seems to operate on stomatal lineages where it controls specific cell-lineage and cell-signaling developmental mechanisms.Ministerio de Ciencia e Innovación (España)Junta de Castilla-La ManchaDepto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEpu