Drought Stress Interacts With Powdery Mildew Infection in Tomato.

Under field conditions, plants are often exposed to more than one stress factor at the same time, and therefore need to adapt to different combinations of stresses. Crosstalk between responses to abiotic and biotic stresses is known to occur, and the interaction between stress responses can be positive or negative. We studied the interaction of drought stress and powdery mildew (PM) infection in tomatoes using near-isogenic tomato lines (NILs) carrying the Ol-1, ol-2, or Ol-4 gene that confers resistance to tomato PM caused by Oidium neolycopersici. Our study demonstrated that drought-induced growth reduction was not further reduced by powdery mildew infection. Drought stress, however, decreased fungal infection in the susceptible genotype Moneymaker (MM) with fungal biomass tending to decrease further as the drought severity increased. Drought stress did not affect PM resistance levels of resistant NIL carrying ol-2 (a mutant of the tomato susceptibility Mlo gene) and Ol-4 an NLR (nucleotide-binding site-LRR) R gene associated with a fast hypersensitivity response (HR) but tended to slightly decrease disease levels of NIL-Ol-1 (no gene characterized yet, associated with a slow HR following PM infection). At the molecular level, genes involved in abscisic acid (ABA), salicylic acid (SA), and ethylene pathways were highly induced under combined stress indicating the involvement of ABA, SA, and ethylene in the crosstalk between abiotic and biotic stress. Messenger RNA expression of the ABA-responsive dehydrin SlTAS14 was induced under drought and combined stress with the highest induction under combined stress, and resistant NIL lines showed higher expression levels than MM. The expression of SlNCED (involved in ABA synthesis) was also upregulated under drought and highly induced under combined stress. Expression levels of pathogen responsive gene SlPR1 (an indicator of the SA pathway) and SlACS (involved in ethylene synthesis) were highly induced under powdery mildew infection in MM and the Ol-1 and were induced the most under combined stress in these lines. Taken together, these findings indicate that drought stress can interact with and influence PM infection in tomatoes in a resistance type-dependent manner. The role of hormonal signaling pathways in the crosstalk between drought stress and PM infection is further discussed.

Whole genome re-sequencing of sweet cherry (Prunus avium L.) yields insights into genomic diversity of a fruit species.

Sweet cherries, Prunus avium L. (Rosaceae), are gaining importance due to their perenniallity and nutritional attributes beneficial for human health. Interestingly, sweet cherry cultivars exhibit a wide range of phenotypic diversity in important agronomic traits, such as flowering time and defense reactions against pathogens. In this study, whole-genome resequencing (WGRS) was employed to characterize genetic variation, population structure and allelic variants in a panel of 20 sweet cherry and one wild cherry genotypes, embodying the majority of cultivated Greek germplasm and a representative of a local wild cherry elite phenotype. The 21 genotypes were sequenced in an average depth of coverage of 33.91×. and effective mapping depth, to the genomic reference sequence of 'Satonishiki' cultivar, between 22.21× to 36.62×. Discriminant analysis of principal components (DAPC) with SNPs revealed two clusters of genotypes. There was a rapid linkage disequilibrium decay, as the majority of SNP pairs with r2 in near complete disequilibrium (>0.8) were found at physical distances less than 10 kb. Functional analysis of the variants showed that the genomic ratio of non-synonymous/synonymous (dN/dS) changes was 1.78. The higher dN frequency in the Greek cohort of sweet cherry could be the result of artificial selection pressure imposed by breeding, in combination with the vegetative propagation of domesticated cultivars through grafting. The majority of SNPs with high impact (e.g., stop codon gaining, frameshift), were identified in genes involved in flowering time, dormancy and defense reactions against pathogens, providing promising resources for future breeding programs. Our study has established the foundation for further large scale characterization of sweet cherry germplasm, enabling breeders to incorporate diverse germplasm and allelic variants to fine tune flowering and maturity time and disease resistance in sweet cherry cultivars.

Whole-genome resequencing of Cucurbita pepo morphotypes to discover genomic variants associated with morphology and horticulturally valuable traits.

Cucurbita pepo contains two cultivated subspecies, each of which encompasses four fruit-shape morphotypes (cultivar groups). The Pumpkin, Vegetable Marrow, Cocozelle, and Zucchini Groups are of subsp. pepo and the Acorn, Crookneck, Scallop, and Straightneck Groups are of subsp. ovifera. Recently, a de novo assembly of the C. pepo subsp. pepo Zucchini genome was published, providing insights into its evolution. To expand our knowledge of evolutionary processes within C. pepo and to identify variants associated with particular morphotypes, we performed whole-genome resequencing of seven of these eight C. pepo morphotypes. We report for the first time whole-genome resequencing of the four subsp. pepo (Pumpkin, Vegetable Marrow, Cocozelle, green Zucchini, and yellow Zucchini) morphotypes and three of the subsp. ovifera (Acorn, Crookneck, and Scallop) morphotypes. A high-depth resequencing approach was followed, using the BGISEQ-500 platform that enables the identification of rare variants, with an average of 33.5X. Approximately 94.5% of the clean reads were mapped against the reference Zucchini genome. In total, 3,823,977 high confidence single-nucleotide polymorphisms (SNPs) were identified. Within each accession, SNPs varied from 636,918 in green Zucchini to 2,656,513 in Crookneck, and were distributed homogeneously along the chromosomes. Clear differences between subspecies pepo and ovifera in genetic variation and linkage disequilibrium are highlighted. In fact, comparison between subspecies pepo and ovifera indicated 5710 genes (22.5%) with Fst > 0.80 and 1059 genes (4.1%) with Fst = 1.00 as potential candidate genes that were fixed during the independent evolution and domestication of the two subspecies. Linkage disequilibrium was greater in subsp. ovifera than in subsp. pepo, perhaps reflective of the earlier differentiation of morphotypes within subsp. ovifera. Some morphotype-specific genes have been localized. Our results offer new clues that may provide an improved understanding of the underlying genomic regions involved in the independent evolution and domestication of the two subspecies. Comparisons among SNPs unique to particular subspecies or morphotypes may provide candidate genes responsible for traits of high economic importance.

Expanding Phaseolus coccineus Genomic Resources: De Novo Transcriptome Assembly and Analysis of Landraces 'Gigantes' and 'Elephantes' Reveals Rich Functional Variation.

Beans are one of the most important staple crops in the world. Runner bean (Phaseolus coccineus L.) is a small-scale agriculture crop compared to common bean (Phaseolusvulgaris). Beans have been introduced to Europe from the Central America to Europe and since then they have been scattered to different geographical regions. This has resulted in the generation of numerous local cultivars and landraces with distinguished characters and adaptive potential. To identify and characterize the underlying genomic variation of two very closely related runner bean cultivars, we performed RNA-Seq with de novo transcriptome assembly in two landraces of P. coccineus, 'Gigantes' and 'Elephantes' phenotypically distinct, differing in seed size and shape. The cleaned reads generated 37,379 and 37,774 transcripts for 'Gigantes' and 'Elephantes,' respectively. A total of 1896 DEGs were identified between the two cultivars, 1248 upregulated in 'Elephantes' and 648 upregulated in 'Gigantes.' A significant upregulation of defense-related genes was observed in 'Elephantes,' among those, numerous members of the AP2-EREBP, WRKY, NAC, and bHLH transcription factor families. In total, 3956 and 4322 SSRs were identified in 'Gigantes' and 'Elephantes,' respectively. Trinucleotide repeats were the most dominant repeat motif, accounting for 41.9% in 'Gigantes' and 40.1% in 'Elephantes' of the SSRs identified, followed by dinucleotide repeats (29.1% in both cultivars). Additionally, 19,281 putative SNPs were identified, among those 3161 were non-synonymous, thus having potential functional implications. High-confidence non-synonymous SNPs were successfully validated with an HRM assay, which can be directly adopted for P. coccineus molecular breeding. These results significantly expand the number of polymorphic markers within P. coccineus genus, enabling the robust identification of runner bean cultivars, the construction of high-resolution genetic maps, potentiating genome-wide association studies. They finally contribute to the genetic reservoir for the improvement of the closely related and intercrossable Phaseolus vulgaris.

The Role of Tomato WRKY Genes in Plant Responses to Combined Abiotic and Biotic Stresses.

In the field, plants constantly face a plethora of abiotic and biotic stresses that can impart detrimental effects on plants. In response to multiple stresses, plants can rapidly reprogram their transcriptome through a tightly regulated and highly dynamic regulatory network where WRKY transcription factors can act as activators or repressors. WRKY transcription factors have diverse biological functions in plants, but most notably are key players in plant responses to biotic and abiotic stresses. In tomato there are 83 WRKY genes identified. Here we review recent progress on functions of these tomato WRKY genes and their homologs in other plant species, such as Arabidopsis and rice, with a special focus on their involvement in responses to abiotic and biotic stresses. In particular, we highlight WRKY genes that play a role in plant responses to a combination of abiotic and biotic stresses.

Plant behaviour under combined stress: tomato responses to combined salinity and pathogen stress.

Crop plants are subjected to a variety of stresses during their lifecycle, including abiotic stress factors such as salinity and biotic stress factors such as pathogens. Plants have developed a multitude of defense and adaptation responses to these stress factors. In the field, different stress factors mostly occur concurrently resulting in a new state of stress, the combined stress. There is evidence that plant resistance to pathogens can be attenuated or enhanced by abiotic stress factors. With stress tolerance research being mostly focused on plant responses to individual stresses, the understanding of a plant's ability to adapt to combined stresses is limited. In the last few years, we studied powdery mildew resistance under salt stress conditions in the model crop plant tomato with the aim to understand the requirements to achieve plant resilience to a wider array of combined abiotic and biotic stress combinations. We uncovered specific responses of tomato plants to combined salinity-pathogen stress, which varied with salinity intensity and plant resistance genes. Moreover, hormones, with their complex regulation and cross-talk, were shown to play a key role in the adaptation of tomato plants to the combined stress. In this review, we attempt to understand the complexity of plant responses to abiotic and biotic stress combinations, with a focus on tomato responses (genetic control and cross-talk of signaling pathways) to combined salinity and pathogen stresses. Further, we provide recommendations on how to design novel strategies for breeding crops with a sustained performance under diverse environmental conditions.

Plant glutathione transferase-mediated stress tolerance: functions and biotechnological applications.

Plant glutathione transferases (EC 2.5.1.18, GSTs) are an ancient, multimember and diverse enzyme class. Plant GSTs have diverse roles in plant development, endogenous metabolism, stress tolerance, and xenobiotic detoxification. Their study embodies both fundamental aspects and agricultural interest, because of their ability to confer tolerance against biotic and abiotic stresses and to detoxify herbicides. Here we review the biotechnological applications of GSTs towards developing plants that are resistant to biotic and abiotic stresses. We integrate recent discoveries, highlight, and critically discuss the underlying biochemical and molecular pathways involved. We elaborate that the functions of GSTs in abiotic and biotic stress adaptation are potentially a result of both catalytic and non-catalytic functions. These include conjugation of reactive electrophile species with glutathione and the modulation of cellular redox status, biosynthesis, binding, and transport of secondary metabolites and hormones. Their major universal functions under stress underline the potential in developing climate-resilient cultivars through a combination of molecular and conventional breeding programs. We propose that future GST engineering efforts through rational and combinatorial approaches, would lead to the design of improved isoenzymes with purpose-designed catalytic activities and novel functional properties. Concurrent GST-GSH metabolic engineering can incrementally increase the effectiveness of GST biotechnological deployment.

Responses to combined abiotic and biotic stress in tomato are governed by stress intensity and resistance mechanism.

Stress conditions in agricultural ecosystems can occur at variable intensities. Different resistance mechanisms against abiotic stress and pathogens are deployed by plants. Thus, it is important to examine plant responses to stress combinations under different scenarios. Here, we evaluated the effect of different levels of salt stress ranging from mild to severe (50, 100, and 150mM NaCl) on powdery mildew resistance and overall performance of tomato introgression lines with contrasting levels of partial resistance, as well as near-isogenic lines (NILs) carrying the resistance gene Ol-1 (associated with a slow hypersensitivity response; HR), ol-2 (an mlo mutant associated with papilla formation), and Ol-4 (an R gene associated with a fast HR). Powdery mildew resistance was affected by salt stress in a genotype- and stress intensity-dependent manner. In susceptible and partial resistant lines, increased susceptibility was observed under mild salt stress (50mM) which was accompanied by accelerated cell death-like senescence. In contrast, severe salt stress (150mM) reduced disease symptoms. Na(+) and Cl(-) accumulation in the leaves was linearly related to the decreased pathogen symptoms under severe stress. In contrast, complete resistance mediated by ol-2 and Ol-4 was unaffected under all treatment combinations, and was associated with a decreased growth penalty. Increased susceptibility and senescence under combined stress in NIL-Ol-1 was associated with the induction of ethylene and jasmonic acid pathway genes and the cell wall invertase gene LIN6. These results highlight the significance of stress severity and resistance type on the plant's performance under the combination of abiotic and biotic stress.

Future-proof crops: challenges and strategies for climate resilience improvement.

Breeding for stress-resilient crops strongly depends on technological and biological advancements that have provided a wealth of information on genetic variants and their contribution to stress tolerance. In the context of the upcoming challenges for agriculture due to climate change, such as prolonged and/or increased stress intensities, CO2 increase and stress combinations, hierarchizing this information is key to accelerating crop improvement towards sustained or even increased productivity. We propose traits with high scalability to yield and crop performance that can be targeted for improvement and provide examples of recent discoveries with potential applicability in breeding. Critical to success is the integrated analysis of the phenotypes of genetic variants across different environmental variables using modelling approaches and high-throughput phenotyping.

Ethylene and Abscisic Acid Signaling Pathways Differentially Influence Tomato Resistance to Combined Powdery Mildew and Salt Stress.

There is currently limited knowledge on the role of hormones in plants responses to combinations of abiotic and pathogen stress factors. This study focused on the response of tomato near-isogenic lines (NILs) that carry the Ol-1, ol-2, and Ol-4 loci, conferring resistance to tomato powdery mildew (PM) caused by Oidium neolycopersici, to combined PM and salt stress. These NILs were crossed with the notabilis (ABA-deficient), defenceless1 (JA-deficient), and epinastic (ET overproducer) tomato mutants to investigate possible roles of hormone signaling in response to combined stresses. In the NILs, marker genes for hormonal pathways showed differential expression patterns upon PM infection. The epinastic mutation resulted in breakdown of resistance in NIL-Ol-1 and NIL-ol-2. This was accompanied by reduced callose deposition, and was more pronounced under combined salt stress. The notabilis mutation resulted in H2O2 overproduction and reduced susceptibility to PM in NIL-Ol-1 under combined stress, but lead to higher plant growth reduction under salinity and combined stress. Resistance in NIL-ol-2 was compromised by the notabilis mutation, which was potentially caused by reduction of callose deposition. Under combined stress the compromised resistance in NIL-ol-2 was restored. PM resistance in NIL-Ol-4 remained robust across all mutant and treatment combinations. Hormone signaling is critical to the response to combined stress and PM, in terms of resistance and plant fitness. ABA appears to be at the crossroads of disease susceptibility/senescence and plant performance under combined stress These gained insights can aid in narrowing down targets for improving crop performance under stress combinations.

Enhancing crop resilience to combined abiotic and biotic stress through the dissection of physiological and molecular crosstalk.

Plants growing in their natural habitats are often challenged simultaneously by multiple stress factors, both abiotic and biotic. Research has so far been limited to responses to individual stresses, and understanding of adaptation to combinatorial stress is limited, but indicative of non-additive interactions. Omics data analysis and functional characterization of individual genes has revealed a convergence of signaling pathways for abiotic and biotic stress adaptation. Taking into account that most data originate from imposition of individual stress factors, this review summarizes these findings in a physiological context, following the pathogenesis timeline and highlighting potential differential interactions occurring between abiotic and biotic stress signaling across the different cellular compartments and at the whole plant level. Potential effects of abiotic stress on resistance components such as extracellular receptor proteins, R-genes and systemic acquired resistance will be elaborated, as well as crosstalk at the levels of hormone, reactive oxygen species, and redox signaling. Breeding targets and strategies are proposed focusing on either manipulation and deployment of individual common regulators such as transcription factors or pyramiding of non- (negatively) interacting components such as R-genes with abiotic stress resistance genes. We propose that dissection of broad spectrum stress tolerance conferred by priming chemicals may provide an insight on stress cross regulation and additional candidate genes for improving crop performance under combined stress. Validation of the proposed strategies in lab and field experiments is a first step toward the goal of achieving tolerance to combinatorial stress in crops.

Overexpression of a specific soybean GmGSTU4 isoenzyme improves diphenyl ether and chloroacetanilide herbicide tolerance of transgenic tobacco plants.

Plant glutathione transferases (GSTs) superfamily consists of multifunctional enzymes and forms a major part of the plants herbicide detoxification enzyme network. The tau class GST isoenzyme GmGSTU4 from soybean, exhibits catalytic activity towards the diphenyl ether herbicide fluorodifen and is active as glutathione-dependent peroxidase (GPOX). Transgenic tobacco plants of Basmas cultivar were generated via Agrobacterium transformation. The aim was to evaluate in planta, GmGSTU4's role in detoxifying the diphenyl ether herbicides fluorodifen and oxyfluorfen and the chloroacetanilides alachlor and metolachlor. Transgenic tobacco plants were verified by PCR and Southern blot hybridization and expression of GmGSTU4 was determined by RT-PCR. Leaf extracts from transgenic plants showed moderate increase in GST activity towards CDNB and a significant increase towards fluorodifen and alachlor, and at the same time an increased GPOX activity towards cumene hydroperoxide. GmGSTU4 overexpressing plants when treated with 200 µM fluorodifen or oxyfluorfen exhibited reduced relative electrolyte leakage compared to wild type plants. Moreover all GmGSTU4 overexpressing lines exhibited significantly increased tolerance towards alachlor when grown in vitro at 7.5 mg/L alachlor compared to wild type plants. No significant increased tolerance was observed to metolachlor. These results confirm the contribution of this particular GmGSTU4 isoenzyme from soybean in the detoxification of fluorodifen and alachlor, and provide the basis towards the development of transgenic plants with improved phytoremediation capabilities for future use in environmental cleanup of herbicides.