While the response of Arabidopsis thaliana to drought, herbivory or fungal infection has been well-examined, the consequences of exposure to a series of such (a)biotic stresses are not well studied. This work reports on the genetic mechanisms underlying the Arabidopsis response to single osmotic stress, and to combinatorial stress, either fungal infection using Botrytis cinerea or herbivory using Pieris rapae caterpillars followed by an osmotic stress treatment. Several small-effect genetic loci associated with rosette dry weight (DW), rosette water content (WC), and the projected rosette leaf area in response to combinatorial stress were mapped using univariate and multi-environment genome-wide association approaches. A single-nucleotide polymorphism (SNP) associated with DROUGHT-INDUCED 19 (DI19) was identified by both approaches, supporting its potential involvement in the response to combinatorial stress. Several SNPs were found to be in linkage disequilibrium with known stress-responsive genes such as PEROXIDASE 34 (PRX34), BASIC LEUCINE ZIPPER 25 (bZIP25), RESISTANCE METHYLATED GENE 1 (RMG1) and WHITE RUST RESISTANCE 4 (WRR4). An antagonistic effect between biotic and osmotic stress was found for prx34 and arf4 mutants, which suggests PRX34 and ARF4 play an important role in the response to the combinatorial stress.

Understanding plant immune responses is complex because of the high interdependence among biological processes in homeostatic networks. Hence, the integration of environmental cues causes network rewiring that interferes with defense responses. Similarly, plants retain molecular signatures configured under abiotic stress periods to rapidly respond to recurrent stress, and these can alter immunity. Metabolome changes imposed by abiotic stressors are persistent, although their impact on defense remains to be clarified. In this study, we profiled metabolomes of Arabidopsis plants under several abiotic stress treatments applied individually or simultaneously to capture temporal trajectories in metabolite composition during adverse conditions and recovery. Further systemic analysis was performed to address the relevance of metabolome changes and extract central features to be tested in planta. Our results demonstrate irreversibility in major fractions of metabolome changes as a general pattern in response to abiotic stress periods. Functional analysis of metabolomes and co-abundance networks points to convergence in the reconfiguration of organic acid and secondary metabolite metabolism. Arabidopsis mutant lines for components related to these metabolic pathways showed altered defense capacities against different pathogens. Collectively, our data suggest that sustained metabolome changes configured in adverse environments can act as modulators of immune responses and provide evidence for a new layer of regulation in plant defense.

BACKGROUND: The extremely harsh environment of the desert is changing dramatically every moment, and the rapid adaptive stress response in the short term requires enormous energy expenditure to mobilize widespread regulatory networks, which is all the more detrimental to the survival of the desert plants themselves. The dune reed, which has adapted to desert environments with complex and variable ecological factors, is an ideal type of plant for studying the molecular mechanisms by which Gramineae plants respond to combinatorial stress of the desert in their natural state. But so far, the data on the genetic resources of reeds is still scarce, therefore most of their research has focused on ecological and physiological studies.
RESULTS: In this study, we obtained the first De novo non-redundant Full-Length Non-Chimeric (FLNC) transcriptome databases for swamp reeds (SR), dune reeds (DR) and the All of Phragmites australis (merged of iso-seq data from SR and DR), using PacBio Iso-Seq technology and combining tools such as Iso-Seq3 and Cogent. We then identified and described long non-coding RNAs (LncRNA), transcription factor (TF) and alternative splicing (AS) events in reeds based on a transcriptome database. Meanwhile, we have identified and developed for the first time a large number of candidates expressed sequence tag-SSR (EST-SSRs) markers in reeds based on UniTransModels. In addition, through differential gene expression analysis of wild-type and homogenous cultures, we found a large number of transcription factors that may be associated with desert stress tolerance in the dune reed, and revealed that members of the Lhc family have an important role in the long-term adaptation of dune reeds to desert environments.
CONCLUSIONS: Our results provide a positive and usable genetic resource for Phragmites australis with a widespread adaptability and resistance, and provide a genetic database for subsequent reeds genome annotation and functional genomic studies.

The growth of 14 Aspergillus strains belonging to nine species was studied under combinatorial deferriprone - H2O2 (iron-chelation - oxidative) stress. When deferriprone pretreated mycelia were subjected to even a weak oxidative stress, the growth inhibitory effect of iron-chelation stress was enhanced in 10 out of 14 strains. In contrast, oxidative stress pretreatment of conidia increased their deferriprone tolerance in 10 strains. Applying iron-chelators as antifungal agent or adjuvant can enhance the efficiency of the combinatorial iron withdrawal - oxidative stress strategy of our immune system and may reduce the survival of conidia escaped from the oxidative attack of pulmonary macrophages.

Nutrient elements play essential roles in plant growth, development, and reproduction. Balanced nutrition is critical for plant health and the ability to withstand biotic stress. Treatment with essential elements has been shown to induce disease resistance in certain cases. Understanding the functional mechanisms underlying plant immune responses to nutritional elements has the potential to provide new insights into crop improvement. In the present study, we investigated the effect of various elements-potassium (K), calcium (Ca), magnesium (Mg), and sodium (Na)-in promoting resistance against the necrotrophic fungus Botrytis cinerea and the hemibiotrophic bacterium Xanthomonas euvesicatoria in tomato. We demonstrate that spray treatment of essential elements was sufficient to activate immune responses, inducing defense gene expression, cellular leakage, reactive oxygen species, and ethylene production. We report that different defense signaling pathways are required for induction of immunity in response to different elements. Our results suggest that genetic mechanisms that are modulated by nutrient elements can be exploited in agricultural practices to promote disease resistance.

In the ever-changing fungal environment, fungi have to cope with a wide array of very different stresses. These stresses frequently act in combination rather than independently, i.e., they quickly follow one another or occur concomitantly. Combinatorial stress response studies revealed that the response of fungi to a stressor is highly dependent on the simultaneous action of other stressors or even on earlier stresses to which the fungi adapted. Several important phenomena were discovered, such as stress pathway interference, acquired stress tolerance, stress response memory or stress cross-protection/sensitization, which cannot be interpreted when we study the consequences of a single stressor alone. Due to the interactions between stressors and stress responses, a stress response that develops under a combined stress is not the simple summation of stress responses observed during single stress treatments. Based on the knowledge collected from single stress treatment experiments, we cannot predict how fungi will respond to a certain combination of stresses or even whether this combination will be more harmful than single stress treatments. This uncertainty warns us that if we want to understand how fungi adapt to a certain habitat (e.g., to the human body) to find a point of weakness in this adaptation, we must understand how the fungi cope with combinations of stresses, rather than with single stressors.

Copper (Cu) and iron (Fe) constitute fundamental nutrients for plant biology but are often limited due to low bioavailability. Unlike responses to single Cu or Fe deprivation, the consequences of simultaneous Cu and Fe deficiency have not yet been fully deciphered. Previously, it was demonstrated that Cu and Fe deficiency applied in combination imposes transcriptome, proteome, and metabolome changes different from those triggered under each deficiency individually. Here, we evaluated the effect of alternative splicing (AS) on the transcriptome of rosette leaves under single and simultaneous Cu and Fe deficiency. Differentially spliced genes (DSGs) and differentially expressed genes (DEGs) coincided in number (2,600 approx.) although the overlapping fraction was minimal (15%). Functional annotation of changes exclusively detected under simultaneous Cu and Fe deficiency revealed that DEGs participated in general stress responses and translation, while DSGs were involved in metabolic reactions, especially amino acid biosynthesis. Interestingly, transcripts encoding central features for tryptophan (Trp) and asparagine (Asn) synthesis - two significantly altered metabolites under simultaneous Cu and Fe deficiency - underwent exclusive intron retention events under the double deficiency. However, transcript and protein amounts for these enzymes did not correlate with Trp and Asn concentration. In consequence, we propose that AS might act as a regulatory mechanism to modify the stability and/or functionality of the enzymes and therefore fine-tune amino acid production during the combinatorial response to simultaneous Cu and Fe deficiency.

The glucocorticoid betamethasone (BM) has potent anti-inflammatory and immunosuppressive effects; however, it increases the susceptibility of patients to superficial Candida infections. Previously we found that this disadvantageous side effect can be counteracted by menadione sodium bisulfite (MSB) induced oxidative stress treatment. The fungus specific protein phosphatase Z1 (CaPpz1) has a pivotal role in oxidative stress response of Candida albicans and was proposed as a potential antifungal drug target. The aim of this study was to investigate the combined effects of CaPPZ1 gene deletion and MSB treatment in BM pre-treated C. albicans cultures. We found that the combined treatment increased redox imbalance, enhanced the specific activities of antioxidant enzymes, and reduced the growth in cappz1 mutant (KO) strain. RNASeq data demonstrated that the presence of BM markedly elevated the number of differentially expressed genes in the MSB treated KO cultures. Accumulation of reactive oxygen species, increased iron content and fatty acid oxidation, as well as the inhibiting ergosterol biosynthesis and RNA metabolic processes explain, at least in part, the fungistatic effect caused by the combined stress exposure. We suggest that the synergism between MSB treatment and CaPpz1 inhibition could be considered in developing of a novel combinatorial antifungal strategy accompanying steroid therapy.

Copper (Cu) and iron (Fe) are essential for plant growth and are often in short supply under natural conditions. Molecular responses to simultaneous lack of both metals (-Cu-Fe) differ from those seen in the absence of either alone. Metabolome profiling of plant leaves previously revealed that fumarate levels fall under -Cu-Fe conditions. We employed lines lacking cytosolic FUMARASE2 (FUM2) activity to study the impact of constitutive suppression of cytosolic fumarate synthesis on plant growth under Cu and/or Fe deficiency. In fum2 mutants, photosynthesis and growth were less impaired under -Cu-Fe conditions than in wild-type (WT) seedlings. In particular, levels of photosynthetic proteins, chloroplast ultrastructure, amino acid profiles and redox state were less perturbed by simultaneous Cu-Fe deficiency in lines that cannot produce fumarate in the cytosol. Although cytosolic fumarate has been reported to promote acclimation of photosynthesis to low temperatures when metal supplies are adequate, the photosynthetic efficiency of fum2 lines grown under Cu-Fe deficiency in the cold was higher than in WT. Uptake and contents of Cu and Fe are similar in WT and fum2 plants under control and -Cu-Fe conditions, and lack of FUM2 does not alter the ability to sense metal deficiency, as indicated by marker gene expression. Collectively, we propose that reduced levels of cytosolic fumarate synthesis ultimately increase the availability of Fe for incorporation into metalloproteins.

Plants have evolved adaptive measures to cope with abiotic and biotic challenges simultaneously. Combinatorial stress responses require environmental signal integration and response prioritization to balance stress adaptation and growth. We have investigated the impact of salt, an important environmental factor in arid regions, on the Arabidopsis innate immune response. Activation of a classical salt stress response resulted in increased susceptibility to infection with hemibiotrophic Pseudomonas syringae or necrotrophic Alternaria brassicicola, and Botrytis cinerea, respectively. Surprisingly, pattern-triggered immunity (PTI)-associated responses were largely unaffected upon salt pre-treatment. However, we further observed a strong increase in phytohormone levels. Particularly, abscisic acid (ABA) levels were already elevated before pathogen infection, and application of exogenous ABA substituted for salt-watering in increasing Arabidopsis susceptibility toward B. cinerea infection. We propose a regulatory role of ABA in attenuating Botrytis immunity in this plant under salt stress conditions.