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The plant VQ motif-containing proteins are a recently discovered class of plant regulatory proteins interacting with WRKY transcription factors capable of modulate their activity as transcriptional regulators. The short VQ motif (FxxhVQxhTG) is the main element in the WRKY-VQ interaction, whereas a newly identified variable upstream amino acid motif appears to be determinant for the WRKY specificity. The VQ family has been studied in several species and seems to play important roles in a variety of biological processes, including response to biotic and abiotic stresses. Here, we present a systematic study of the VQ family in both diploid (Fragaria vesca) and octoploid (Fragaria x ananassa) strawberry species. Thus, twenty-five VQ-encoding genes were identified and twenty-three were further confirmed by gene expression analysis in different tissues and fruit ripening stages. Their expression profiles were also studied in F. ananassa fruits affected by anthracnose, caused by the ascomycete fungus Colletotrichum, a major pathogen of strawberry, and in response to the phytohormones salicylic acid and methyl-jasmonate, which are well established as central stress signals to regulate defence responses to pathogens. This comprehensive analysis sheds light for a better understanding of putative implications of members of the VQ family in the defence mechanisms against this major pathogen in strawberry.

WRKY transcription factors play critical roles in plant growth and development or stress responses. Using up-to-date genomic data, a total of 64 and 257 WRKY genes have been identified in the diploid woodland strawberry, Fragaria vesca, and the more complex allo-octoploid commercial strawberry, Fragaria × ananassa cv. Camarosa, respectively. The completeness of the new genomes and annotations has enabled us to perform a more detailed evolutionary and functional study of the strawberry WRKY family members, particularly in the case of the cultivated hybrid, in which homoeologous and paralogous FaWRKY genes have been characterized. Analysis of the available expression profiles has revealed that many strawberry WRKY genes show preferential or tissue-specific expression. Furthermore, significant differential expression of several FaWRKY genes has been clearly detected in fruit receptacles and achenes during the ripening process and pathogen challenged, supporting a precise functional role of these strawberry genes in such processes. Further, an extensive analysis of predicted development, stress and hormone-responsive cis-acting elements in the strawberry WRKY family is shown. Our results provide a deeper and more comprehensive knowledge of the WRKY gene family in strawberry.

Strawberry (Fragaria ×ananassa) is a major food crop worldwide, due to the flavor, aroma and health benefits of the fruit, but its productivity and quality are seriously limited by a large variety of phytopathogens, including Colletotrichum spp. So far, key factors regulating strawberry immune response remain unknown. The FaWRKY1 gene has been previously proposed as an important element mediating defense responses in strawberry to Colletotrichum acutatum. To get further insight into the functional role that FaWRKY1 plays in the defense mechanism, Agrobacterium-mediated transient transformation was used both to silence and overexpress the FaWRKY1 gene in strawberry fruits (Fragaria ×ananassa cv. Primoris), which were later analyzed upon C. acutatum inoculation. Susceptibility tests were performed after pathogen infection comparing the severity of disease between the two agroinfiltrated opposite halves of the same fruit, one half bearing a construct either for FaWRKY1 overexpression or RNAi-mediated silencing and the other half bearing the empty vector, as control. The severity of tissue damage was monitored and found to be visibly reduced at five days after pathogen inoculation in the fruit half where FaWRKY1 was transiently silenced compared to that of the opposite control half and statistical analysis corroborated a significant reduction in disease susceptibility. Contrarily, a similar level of susceptibility was found when FaWRKY1 overexpression and control fruit samples, was compared. These results unravel a negative regulatory role of FaWRKY1 in resistance to the phytopathogenic fungus C. acutatum in strawberry fruit and contrast with the previous role described for this gene in Arabidopsis as positive regulator of resistance against the bacteria Pseudomonas syringae. Based on previous results, a tentative working model for WRKY75 like genes after pathogen infection is proposed and the expression pattern of potential downstream FaWRKY1 target genes was also analyzed in strawberry fruit upon C. acutatum infection. Our results highlight that FaWRKY1 might display different function according to species, plant tissue and/or type of pathogen and underline the intricate FaWRKY1 responsive defense regulatory mechanism taking place in strawberry against this important crop pathogen., This work was supported by Junta de Andalucía, Spain (Proyectos de Excelencia P12-AGR2174, and grants to Grupo-BIO278).

Strawberry (

Natural substances such as elicitors of plant defense response, has become a promising option for effective management of plant diseases and are a prerequisite for sustainable and ecological agriculture. Recognition of diverse elicitors, effectors and modulators by specific and nonspecific plant receptors activates signalling cascades eventually leading to gene expression and defense responses. Elicited plants or "primed plants" display either faster, stronger, or both activation of the various cellular defense responses that are induced following attack by either pathogens or insects or in response to abiotic stress. The aim of this work has been to develop new elicitor compounds from natural vegetal substrates, and predict its effectiveness in the field by using robust molecular techniques and appropriate biomarkers of plant defense. Thus, Fragaria x ananassa Duch. cv. Chandler cell suspension cultures, and real-time, quantitative PCR analysis (RTqPCR) were utilized to test the elicitor capability of new natural elicitors. This experimental approach was applied to analyze the pattern of gene expression of specific strawberry genes already known to be good markers of SA- and JA-dependent signalling pathways, which were used as defense induced biomarkers in plants. In addition, determination of total phenols and antioxidant activity, and HPLC-DAD-MS analysis of polyphenolic compounds was performed in these cell suspension samples. Susceptibility to C. acutatum of strawberry plants treated with the new elicitor was performed in greenhouse under controlled conditions. In collaboration with AGROMETODOS, S.A., we have produced new natural compounds that can act as elicitors. We have compared the elicitor activity of a new compound (EH) with the commercial product BROTOMAX ® . The new product EH and BROTOMAX ® activate plant defense related genes in cell suspension cultures. Also, both products induce polyphenol production in this assay. The new product EH and BROTOMAX ® increased the strawberry (Fragaria x ananassa Duch. cv. Camarosa) and olive (Olea europaea L. cv. Picual) resistance against Colletotrichum acutatum and Fusicladium oleaginum plant infections, respectively. We can conclude that new natural bioelicitors can be produced by low-cost procedures based on fermentation of natural substrates derived from plants. The use of suitable, sensitive, appropriate and robust molecular biomarkers also provide an excellent molecular tool to predict the effectiveness in the field of natural compounds with potential eliciting ability, which may be conveniently exploited as ecological bioinducers of plant defense response in strawberry and in many other important crops.

Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen., This work was supported by Junta de Andalucía, Spain [Proyectos de Excelencia P07-AGR-02482/P12-AGR-2174, and grants to Grupo-BIO278].

Strawberry (Fragaria spp) is an emerging model for the development of basic genomics and recombinant DNA studies among rosaceous crops. Functional genomic and molecular studies involve relative quantification of gene expression under experimental conditions of interest. Accuracy and reliability are dependent upon the choice of an optimal reference control transcript. There is no information available on validated endogenous reference genes for use in studies testing strawberrypathogen interactions. Thirteen potential pre-selected strawberry reference genes were tested against different tissues, strawberry cultivars, biotic stresses, ripening and senescent conditions, and SA/JA treatments. Evaluation of reference candidate’s suitability was analyzed by five different methodologies, and information was merged to identify best reference transcripts. A combination of all five methods was used for selective classification of reference genes. The resulting superior reference genes, FaRIB413, FaACTIN, FaEF1a and FaGAPDH2 are strongly recommended as control genes for relative quantification of gene expression in strawberry. This report constitutes the first systematic study to identify and validate optimal reference genes for accurate normalization of gene expression in strawberry plant defense response studies.