FaMYB123 interacts with FabHLH3 to regulate the late steps of anthocyanin and flavonol biosynthesis during ripening.

In this work, we identified and functionally characterized the strawberry (Fragaria × ananassa) R2R3 MYB transcription factor FaMYB123. As in most genes associated with organoleptic properties of ripe fruit, FaMYB123 expression is ripening-related, receptacle-specific, and antagonistically regulated by ABA and auxin. Knockdown of FaMYB123 expression by RNAi in ripe strawberry fruit receptacles downregulated the expression of enzymes involved in the late steps of anthocyanin/flavonoid biosynthesis. Transgenic fruits showed a parallel decrease in the contents of total anthocyanin and flavonoid, especially malonyl derivatives of pelargonidin and cyanidins. The decrease was concomitant with accumulation of proanthocyanin, propelargonidins, and other condensed tannins associated mainly with green receptacles. Potential coregulation between FaMYB123 and FaMYB10, which may act on different sets of genes for the enzymes involved in anthocyanin production, was explored. FaMYB123 and FabHLH3 were found to interact and to be involved in the transcriptional activation of FaMT1, a gene responsible for the malonylation of anthocyanin components during ripening. Taken together, these results demonstrate that FaMYB123 regulates the late steps of the flavonoid pathway in a specific manner. In this study, a new function for an R2R3 MYB transcription factor, regulating the expression of a gene that encodes a malonyltransferase, has been elucidated.

Elucidating the role of polygalacturonase genes in strawberry fruit softening.

To disentangle the role of polygalacturonase (PG) genes in strawberry softening, the two PG genes most expressed in ripe receptacles, FaPG1 and FaPG2, were down-regulated. Transgenic ripe fruits were firmer than those of the wild type when PG genes were silenced individually. Simultaneous silencing of both PG genes by transgene stacking did not result in an additional increase in firmness. Cell walls from ripe fruits were characterized by a carbohydrate microarray. Higher signals of homogalacturonan and rhamnogalacturonan I pectin epitopes in polysaccharide fractions tightly bound to the cell wall were observed in the transgenic genotypes, suggesting a lower pectin solubilization. At the transcriptomic level, the suppression of FaPG1 or FaPG2 alone induced few transcriptomic changes in the ripe receptacle, but the amount of differentially expressed genes increased notably when both genes were silenced. Many genes encoding cell wall-modifying enzymes were down-regulated. The expression of a putative high affinity potassium transporter was induced in all transgenic genotypes, indicating that cell wall weakening and loss of cell turgor could be linked. These results suggest that, besides the disassembly of pectins tightly linked to the cell wall, PGs could play other roles in strawberry softening, such as the release of oligogalacturonides exerting a positive feedback in softening.

An atypical HLH transcriptional regulator plays a novel and important role in strawberry ripened receptacle.

BACKGROUND: In soft fruits, the differential expression of many genes during development and ripening is responsible for changing their organoleptic properties. In strawberry fruit, although some genes involved in the metabolic regulation of the ripening process have been functionally characterized, some of the most studied genes correspond to transcription factors. High throughput transcriptomics analyses performed in strawberry red receptacle (Fragaria x ananassa) allowed us to identify a ripening-related gene that codes an atypical HLH (FaPRE1) with high sequence homology with the PACLOBUTRAZOL RESISTANCE (PRE) genes. PRE genes are atypical bHLH proteins characterized by the lack of a DNA-binding domain and whose function has been linked to the regulation of cell elongation processes. RESULTS: FaPRE1 sequence analysis indicates that this gene belongs to the subfamily of atypical bHLHs that also includes ILI-1 from rice, SlPRE2 from tomato and AtPRE1 from Arabidopsis, which are involved in transcriptional regulatory processes as repressors, through the blockage by heterodimerization of bHLH transcription factors. FaPRE1 presented a transcriptional model characteristic of a ripening-related gene with receptacle-specific expression, being repressed by auxins and activated by abscisic acid (ABA). However, its expression was not affected by gibberellic acid (GA3). On the other hand, the transitory silencing of FaPRE1 transcription by agroinfiltration in receptacle produced the down-regulation of a group of genes related to the ripening process while inducing the transcription of genes involved in receptacle growth and development. CONCLUSIONS: In summary, this work presents for the first time experimental data that support an important novel function for the atypical HLH FaPRE1 during the strawberry fruit ripening. We hypothesize that FaPRE1 modulates antagonistically the transcription of genes related to both receptacle growth and ripening. Thus, FaPRE1 would repress the expression of receptacle growth promoting genes in the ripened receptacle, while it would activate the expression of those genes related to the receptacle ripening process.

Antisense down-regulation of the strawberry ß-galactosidase gene FaßGal4 increases cell wall galactose levels and reduces fruit softening.

Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by ß-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative ß-galactosidase gene, FaßGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaßGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaßGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaßGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaßGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaßGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness.

Expression of the ß-1,3-glucanase gene bgn13.1 from Trichoderma harzianum in strawberry increases tolerance to crown rot diseases but interferes with plant growth.

The expression of antifungal genes from Trichoderma harzianum, mainly chitinases, has been used to confer plant resistance to fungal diseases. However, the biotechnological potential of glucanase genes from Trichoderma has been scarcely assessed. In this research, transgenic strawberry plants expressing the ß-1,3-glucanase gene bgn13.1 from T. harzianum, under the control of the CaMV35S promoter, have been generated. After acclimatization, five out of 12 independent lines analysed showed a stunted phenotype when growing in the greenhouse. Moreover, most of the lines displayed a reduced yield due to both a reduction in the number of fruit per plant and a lower fruit size. Several transgenic lines showing higher glucanase activity in leaves than control plants were selected for pathogenicity tests. When inoculated with Colletotrichum acutatum, one of the most important strawberry pathogens, transgenic lines showed lower anthracnose symptoms in leaf and crown than control. In the three lines selected, the percentage of plants showing anthracnose symptoms in crown decreased from 61 % to a mean value of 16.5 %, in control and transgenic lines, respectively. Some transgenic lines also showed an enhanced resistance to Rosellinia necatrix, a soil-borne pathogen causing root and crown rot in strawberry. These results indicate that bgn13.1 from T. harzianum can be used to increase strawberry tolerance to crown rot diseases, although its constitutive expression affects plant growth and fruit yield. Alternative strategies such as the use of tissue specific promoters might avoid the negative effects of bgn13.1 expression in plant performance.

Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy.

BACKGROUND: One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening. SCOPE: In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected.

Effect of heterologous expression of FT gene from Medicago truncatula in growth and flowering behavior of olive plants.

Olive (Olea europaea L. subsp. europaea) is one of the most important crops of the Mediterranean Basin and temperate areas worldwide. Obtaining new olive varieties adapted to climatic changing conditions and to modern agricultural practices, as well as other traits such as biotic and abiotic stress resistance and increased oil quality, is currently required; however, the long juvenile phase, as in most woody plants, is the bottleneck in olive breeding programs. Overexpression of genes encoding the 'florigen' Flowering Locus T (FT), can cause the loss of the juvenile phase in many perennials including olives. In this investigation, further characterization of three transgenic olive lines containing an FT encoding gene from Medicago truncatula, MtFTa1, under the 35S CaMV promoter, was carried out. While all three lines flowered under in vitro conditions, one of the lines stopped flowering after acclimatisation. In soil, all three lines exhibited a modified plant architecture; e.g., a continuous branching behaviour and a dwarfing growth habit. Gene expression and hormone content in shoot tips, containing the meristems from which this phenotype emerged, were examined. Higher levels of OeTFL1, a gene encoding the flowering repressor TERMINAL FLOWER 1, correlated with lack of flowering. The branching phenotype correlated with higher content of salicylic acid, indole-3-acetic acid and isopentenyl adenosine, and lower content of abscisic acid. The results obtained confirm that heterologous expression of MtFTa1 in olive induced continuous flowering independently of environmental factors, but also modified plant architecture. These phenotypical changes could be related to the altered hormonal content in transgenic plants.

Suppressing the rhamnogalacturonan lyase gene FaRGLyase1 preserves RGI pectin degradation and enhances strawberry fruit firmness.

Plant rhamnogalacturonan lyases (RGLyases) cleave the backbone of rhamnogalacturonan I (RGI), the "hairy" pectin and polymer of the disaccharide rhamnose (Rha)-galacturonic acid (GalA) with arabinan, galactan or arabinogalactan side chains. It has been suggested that RGLyases could participate in remodeling cell walls during fruit softening, but clear evidence has not been reported. To investigate the role of RGLyases in strawberry softening, a genome-wide analysis of RGLyase genes in the genus Fragaria was performed. Seventeen genes encoding RGLyases with functional domains were identified in Fragaria × ananassa. FaRGLyase1 was the most expressed in the ripe receptacle of cv. Chandler. Transgenic strawberry plants expressing an RNAi sequence of FaRGLyase1 were obtained. Three transgenic lines yielded ripe fruits firmer than controls without other fruit quality parameters being significantly affected. The highest increase in firmness achieved was close to 32%. Cell walls were isolated from ripe fruits of two selected lines. The amount of water-soluble and chelated pectins was higher in transgenic lines than in the control. A carbohydrate microarray study showed a higher abundance of RGI epitopes in pectin fractions and in the cellulose-enriched fraction obtained from transgenic lines. Sixty-seven genes were differentially expressed in transgenic ripe fruits when compared with controls. These genes were involved in various physiological processes, including cell wall remodeling, ion homeostasis, lipid metabolism, protein degradation, stress response, and defense. The transcriptomic changes observed in FaRGLyase1 plants suggest that senescence was delayed in transgenic fruits.

Insights into the effects of polygalacturonase FaPG1 gene silencing on pectin matrix disassembly, enhanced tissue integrity, and firmness in ripe strawberry fruits.

Antisense-mediated down-regulation of the fruit-specific polygalacturonase (PG) gene FaPG1 in strawberries (Fragaria×ananassa Duch.) has been previously demonstrated to reduce fruit softening and to extend post-harvest shelf life, despite the low PG activity detected in this fruit. The improved fruit traits were suggested to be attributable to a reduced cell wall disassembly due to FaPG1 silencing. This research provides empirical evidence that supports this assumption at the biochemical, cellular, and tissue levels. Cell wall modifications of two independent transgenic antisense lines that demonstrated a >90% reduction in FaPG1 transcript levels were analysed. Sequential extraction of cell wall fractions from control and ripe fruits exhibited a 42% decrease in pectin solubilization in transgenic fruits. A detailed chromatographic analysis of the gel filtration pectin profiles of the different cell wall fractions revealed a diminished depolymerization of the more tightly bound pectins in transgenic fruits, which were solubilized with both a chelating agent and sodium carbonate. The cell wall extracts from antisense FaPG1 fruits also displayed less severe in vitro swelling. A histological analysis revealed more extended cell-cell adhesion areas and an enhanced tissue integrity in transgenic ripe fruits. An immunohistological analysis of fruit sections using the JIM5 antibody against low methyl-esterified pectins demonstrated a higher labelling in transgenic fruit sections, whereas minor differences were observed with JIM7, an antibody that recognizes highly methyl-esterified pectins. These results support that the increased firmness of transgenic antisense FaPG1 strawberry fruits is predominantly due to a decrease in pectin solubilization and depolymerization that correlates with more tightly attached cell wall-bound pectins. This limited disassembly in the transgenic lines indicates that these pectin fractions could play a key role in tissue integrity maintenance that results in firmer ripe fruit.

The strawberry (Fragariaxananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae.

Pectins are essential components of primary plant cell walls and middle lamellae, and are related to the consistency of the fruit and its textural changes during ripening. In fact, strawberries become soft as the middle lamellae of cortical parenchyma cells are extensively degraded during ripening, leading to the observed short post-harvest shelf life. Using a custom-made oligonucleotide-based strawberry microarray platform, a putative rhamnogalacturonate lyase gene (FaRGlyase1) was identified. Bioinformatic analysis of the FaRGlyase1 sequence allowed the identification of a conserved rhamnogalacturonate lyase domain, which was also present in other putative RGlyase sequences deposited in the databases. Expression of FaRGlyase1 occurred mainly in the receptacle, concurrently with ripening, and it was positively regulated by abscisic acid and negatively by auxins. FaRGLyase1 gene expression was transiently silenced by injecting live Agrobacterium cells harbouring RNA interference constructs into fruit receptacles. Light and electron microscopy analyses of these transiently silenced fruits revealed that this gene is involved in the degradation of pectins present in the middle lamella region between parenchymatic cells. In addition, genetic linkage association analyses in a strawberry-segregating population showed that FaRGLyase1 is linked to a quantitative trait loci linkage group related to fruit hardness and firmness. The results showed that FaRGlyase1 could play an important role in the fruit ripening-related softening process that reduces strawberry firmness and post-harvest life.

CRISPR/Cas9 editing of the polygalacturonase FaPG1 gene improves strawberry fruit firmness.

Firmness is one of the most important fruit quality traits in strawberries. The postharvest shelf life of this soft fruit is highly limited by the loss of firmness, where cell wall disassembly plays an important role. Previous studies demonstrated that the polygalacturonase FaPG1 has a key role in remodelling pectins during strawberry softening. In this study, FaPG1 knockout strawberry plants have been generated using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Ten independent lines, cv. "Chandler", were obtained, and all of them were successfully edited as determined by PCR amplification and T7 endonuclease assay. The targeted mutagenesis insertion and deletion rates were analyzed using targeted deep sequencing. The percentage of edited sequences varied from 47% up to almost 100%, being higher than 95% for seven of the selected lines. Phenotypic analyses showed that 7 out of the eight lines analyzed produced fruits significantly firmer than the control, ranging from 33 to 70% increase in firmness. There was a positive relationship between the degree of FaPG1 editing and the rise in fruit firmness. Minor changes were observed in other fruit quality traits, such as colour, soluble solids, titratable acidity or anthocyanin content. Edited fruits showed a reduced softening rate during postharvest, displayed a reduced transpirational water loss, and were less damaged by Botrytis cinerea inoculation. The analysis of four potential off-target sites revealed no mutation events. In conclusion, editing the FaPG1 gene using the CRISPR/Cas9 system is an efficient method for improving strawberry fruit firmness and shelf life.

Olive (Olea europaea L.) Genetic Transformation: Current Status and Future Prospects.

Olive (Olea europaea L.) is the most characteristic and important oil crop of the Mediterranean region. Traditional olive cultivation is based on few tens cultivars of ancient origin. To improve this crop, novel selections with higher tolerance to biotic and abiotic stress, adaptable to high-density planting systems and resilient to climate change are needed; however, breeding programs are hindered by the long juvenile period of this species and few improved genotypes have been released so far. Genetic transformation could be of great value, in the near future, to develop new varieties or rootstocks in a shorter time; in addition, it has currently become an essential tool for functional genomic studies. The recalcitrance of olive tissues to their in vitro manipulation has been the main bottleneck in the development of genetic transformation procedures in this species; however, some important traits such as fungal resistance, flowering or lipid composition have successfully been manipulated through the genetic transformation of somatic embryos of juvenile or adult origin, providing a proof of the potential role that this technology could have in olive improvement. However, the optimization of these protocols for explants of adult origin is a prerequisite to obtain useful materials for the olive industry. In this review, initially, factors affecting plant regeneration via somatic embryogenesis are discussed. Subsequently, the different transformation approaches explored in olive are reviewed. Finally, transgenic experiments with genes of interest undertaken to manipulate selected traits are discussed.

Modification of 13-hydroperoxide lyase expression in olive affects plant growth and results in altered volatile profile.

The C6 aldehydes, alcohols, and the corresponding esters are the most important compounds of virgin olive oil aroma. These C6 volatile compounds are synthesized via the 13-hydroperoxide lyase (13-HPL) branch of the lipoxygenase pathway. In this investigation, a functional analysis of the olive (Olea europaea L.) 13-HPL gene by its overexpression and silencing in olive transgenic lines was carried out. With this aim, sense and RNAi constructs of the olive 13-HPL gene were generated and used for the transformation of embryogenic olive cultures. Leaves from overexpressing lines showed a slight increase in 13-HPL gene expression, whereas RNAi lines exhibited a strong decrease in their transcript levels. Quantification of 13-HPL activity in two overexpressing and two RNAi lines showed a positive correlation with levels of transcripts. Interestingly, RNAi lines showed a high decrease in the content of C6 volatiles linked to a strong increase of C5 volatile compounds, altering the volatile profile in the leaves. In addition, the silencing of the 13-HPL gene severely affected plant growth and development. This investigation demonstrates the role of the 13-HPL gene in the biogenesis of olive volatile compounds and constitutes a functional genomics study in olive related to virgin olive oil quality.

Heterologous Expression of the AtNPR1 Gene in Olive and Its Effects on Fungal Tolerance.

The NPR1 gene encodes a key component of systemic acquired resistance (SAR) signaling mediated by salicylic acid (SA). Overexpression of NPR1 confers resistance to biotrophic and hemibiotrophic fungi in several plant species. The NPR1 gene has also been shown to be involved in the crosstalk between SAR signaling and the jasmonic acid-ethylene (JA/Et) pathway, which is involved in the response to necrotrophic fungi. The aim of this research was to generate transgenic olive plants expressing the NPR1 gene from Arabidopsis thaliana to evaluate their differential response to the hemibiotrophic fungus Verticillium dahliae and the necrotroph Rosellinia necatrix. Three transgenic lines expressing the AtNPR1 gene under the control of the constitutive promoter CaMV35S were obtained using an embryogenic line derived from a seed of cv. Picual. After maturation and germination of the transgenic somatic embryos, the plants were micropropagated and acclimated to ex vitro conditions. The level of AtNPR1 expression in the transgenic materials varied greatly among the different lines and was higher in the NPR1-780 line. The expression of AtNPR1 did not alter the growth of transgenic plants either in vitro or in the greenhouse. Different levels of transgene expression also did not affect basal endochitinase activity in the leaves, which was similar to that of control plants. Response to the hemibiotrophic pathogen V. dahliae varied with pathotype. All plants died by 50 days after inoculation with defoliating (D) pathotype V-138, but the response to non-defoliating (ND) strains differed by race: following inoculation with the V-1242 strain (ND, race 2), symptoms appeared after 44-55 days, with line NPR1-780 showing the lowest disease severity index. This line also showed good performance when inoculated with the V-1558 strain (ND, race 1), although the differences from the control were not statistically significant. In response to the necrotroph R. necatrix, all the transgenic lines showed a slight delay in disease development, with mean area under the disease progress curve (AUDPC) values 7-15% lower than that of the control.

Exploring the Use of Fruit Callus Culture as a Model System to Study Color Development and Cell Wall Remodeling during Strawberry Fruit Ripening.

Cell cultures derived from strawberry fruit at different developmental stages have been obtained to evaluate their potential use to study different aspects of strawberry ripening. Callus from leaf and cortical tissue of unripe-green, white, and mature-red strawberry fruits were induced in a medium supplemented with 11.3 µM 2,4-dichlorophenoxyacetic acid (2,4-D) under darkness. The transfer of the established callus from darkness to light induced the production of anthocyanin. The replacement of 2,4-D by abscisic acid (ABA) noticeably increased anthocyanin accumulation in green-fruit callus. Cell walls were isolated from the different fruit cell lines and from fruit receptacles at equivalent developmental stages and sequentially fractionated to obtain fractions enriched in soluble pectins, ester bound pectins, xyloglucans (XG), and matrix glycans tightly associated with cellulose microfibrils. These fractions were analyzed by cell wall carbohydrate microarrays. In fruit receptacle samples, pectins were abundant in all fractions, including those enriched in matrix glycans. The amount of pectin increased from green to white stage, and later these carbohydrates were solubilized in red fruit. Apparently, XG content was similar in white and red fruit, but the proportion of galactosylated XG increased in red fruit. Cell wall fractions from callus cultures were enriched in extensin and displayed a minor amount of pectins. Stronger signals of extensin Abs were detected in sodium carbonate fraction, suggesting that these proteins could be linked to pectins. Overall, the results obtained suggest that fruit cell lines could be used to analyze hormonal regulation of color development in strawberry but that the cell wall remodeling process associated with fruit softening might be masked by the high presence of extensin in callus cultures.

Plant Regeneration via Somatic Embryogenesis in Mature Wild Olive Genotypes Resistant to the Defoliating Pathotype of Verticillium dahliae.

Regeneration capacity, via somatic embryogenesis, of four wild olive genotypes differing in their response to defoliating Verticillium dahliae (resistant genotypes StopVert, OutVert, Ac-18 and the susceptible one, Ac-15) has been evaluated. To induce somatic embryogenesis, methodologies previously used in wild or cultivated olive were used. Results revealed the importance of genotype, explant type, and hormonal balance in the induction process. Use of apical buds obtained from micropropagated shoots following a methodology used in cultivated olive (4 days induction in liquid 1/2 MS medium supplemented with 30 µM TDZ-0.54 µM NAA, followed by 8 weeks in basal 1/2 MS medium) was adequate to obtain somatic embryos in two genotypes, StopVert and Ac-18, with a 5.0 and 2.5% induction rates, respectively; however, no embryogenic response was observed in the other two genotypes. Embryogenic cultures were transferred to basal ECO medium supplemented with 0.5 µM 2iP, 0.44 µM BA, and 0.25 µM indole-3-butyric acid (IBA) for further proliferation. Somatic embryos from StopVert were maturated and germinated achieving a 35.4% conversion rate. An analysis of genetic stability on StopVert, using Simple Sequence Repeats (SSRs) and Random Amplified Polymorphic DNA (RAPDs) markers, was carried out in embryogenic callus, plants regenerated from this callus and two controls, micropropagated shoots used as explant source, and the original mother plant. Polymorphism was only observed in the banding pattern generated by RAPDs in 1 of the 10 callus samples evaluated, resulting in a variation rate of 0.07%. This is the first time in which plants have been regenerated via somatic embryogenesis in wild olive.

The Strawberry FaWRKY1 Transcription Factor Negatively Regulates Resistance to Colletotrichum acutatum in Fruit Upon Infection.

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.

Antisense inhibition of a pectate lyase gene supports a role for pectin depolymerization in strawberry fruit softening.

Cell wall disassembly in softening fruits is a complex process involving the cumulative action of many families of wall-modifying proteins on interconnected polysaccharide matrices. One strategy to elucidate the in vivo substrates of specific enzymes and their relative importance and contribution to wall modification is to suppress their expression in transgenic fruit. It has been reported previously that inhibiting the expression of pectate lyase genes by antisense technology in strawberry (Fragaria x ananassa Duch.) fruit resulted in prolonged fruit firmness. This suggested that pectin depolymerization might make a more important contribution to strawberry fruit softening than is often stated. In this present study, three independent transgenic lines were identified exhibiting a greater than 90% reduction in pectate lyase transcript abundance. Analyses of sequential cell wall extracts from the transgenic and control fruit collectively showed clear quantitative and qualitative differences in the extractability and molecular masses of populations of pectin polymers. Wall extracts from transgenic fruits showed a reduction in pectin solubility and decreased depolymerization of more tightly bound polyuronides. Additional patterns of differential extraction of other wall-associated pectin subclasses were apparent, particularly in the sodium carbonate- and chelator-soluble polymers. In addition, microscopic studies revealed that the typical ripening-associated loss of cell-cell adhesion was substantially reduced in the transgenic fruits. These results indicate that pectate lyase plays an important degradative role in the primary wall and middle lamella in ripening strawberry fruit, and should be included in synergistic models of cell wall disassembly.

Usage of the Heterologous Expression of the Antimicrobial Gene afp From Aspergillus giganteus for Increasing Fungal Resistance in Olive.

The antifungal protein (AFP) produced by Aspergillus giganteus, encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix, was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. 'Picual' were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae.

Unravelling the nanostructure of strawberry fruit pectins by endo-polygalacturonase digestion and atomic force microscopy.

Pectins analysed by AFM are visualized as individual chains, branched or unbranched, and aggregates. To investigate the nature of these structures, sodium carbonate soluble pectins from strawberry fruits were digested with endo-polygalacturonase M2 from Aspergillus aculeatus and visualized by AFM. A gradual decrease in the length of chains was observed as result of the treatment, reaching a minimum LN value of 22nm. The branches were not visible after 2h of enzymatic incubation. The size of complexes also diminished significantly with the enzymatic digestion. A treatment to hydrolyse rhamnogalacturonan II borate diester bonds neither affected chains length or branching nor complex size but reduced the density of aggregates. These results suggest that chains are formed by a mixture of homogalacturonan and more complex molecules composed by a homogalacturonan unit linked to an endo-PG resistant unit. Homogalacturonan is a structural component of the complexes and rhamnogalacturonan II could be involved in their formation.