Editing melon eIF4E associates with virus resistance and male sterility.

The cap-binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap-dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single-nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non-transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non-mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild-type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild-type. An RNA-Seq analysis identified critical genes in pollen development that were down-regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E-specific mRNA translation initiation is a limiting factor for male gametes formation in melon.

Transcriptomic profiling of Melon necrotic spot virus-infected melon plants revealed virus strain and plant cultivar-specific alterations.

BACKGROUND: Viruses are among the most destructive and difficult to control plant pathogens. Melon (Cucumis melo L.) has become the model species for the agriculturally important Cucurbitaceae family. Approaches that take advantage of recently developed genomic tools in melon have been extremely useful for understanding viral pathogenesis and can contribute to the identification of target genes for breeding new resistant cultivars. In this work, we have used a recently described melon microarray for transcriptome profiling of two melon cultivars infected with two strains of Melon necrotic spot virus (MNSV) that only differ on their 3'-untranslated regions. RESULTS: Melon plant tissues from the cultivars Tendral or Planters Jumbo were locally infected with either MNSV-Mα5 or MNSV-Mα5/3'264 and analysed in a time-course experiment. Principal component and hierarchical clustering analyses identified treatment (healthy vs. infected) and sampling date (3 vs. 5 dpi) as the primary and secondary variables, respectively. Out of 7566 and 7074 genes deregulated by MNSV-Mα5 and MNSV-Mα5/3'264, 1851 and 1356, respectively, were strain-specific. Likewise, MNSV-Mα5/3'264 specifically deregulated 2925 and 1618 genes in Tendral and Planters Jumbo, respectively. The GO categories that were significantly affected were clearly different for the different virus/host combinations. Grouping genes according to their patterns of expression allowed for the identification of two groups that were specifically deregulated by MNSV-Mα5/3'264 with respect to MNSV-Mα5 in Tendral, and one group that was antagonistically regulated in Planters Jumbo vs. Tendral after MNSV-Mα5/3'264 infection. Genes in these three groups belonged to diverse functional classes, and no obvious regulatory commonalities were identified. When data on MNSV-Mα5/Tendral infections were compared to equivalent data on cucumber mosaic virus or watermelon mosaic virus infections, cytokinin-O-glucosyltransferase2 was identified as the only gene that was deregulated by all three viruses, with infection dynamics correlating with the amplitude of transcriptome remodeling. CONCLUSIONS: Strain-specific changes, as well as cultivar-specific changes, were identified by profiling the transcriptomes of plants from two melon cultivars infected with two MNSV strains. No obvious regulatory features shared among deregulated genes have been identified, pointing toward regulation through differential functional pathways.

Pepino mosaic virus RNA-Dependent RNA Polymerase POL Domain Is a Hypersensitive Response-Like Elicitor Shared by Necrotic and Mild Isolates.

Pepino mosaic virus (PepMV) is an emerging pathogen that represents a serious threat to tomato production worldwide. PepMV-induced diseases manifest with a wide range of symptoms, including systemic necrosis. Our results showed that PepMV accumulation depends on the virus isolate, tomato cultivar, and environmental conditions, and associates with the development of necrosis. Substitution of lysine for glutamic acid at position 67 in the triple gene block 3 (TGB3) protein, previously described as a necrosis determinant, led to increased virus accumulation and was necessary but not sufficient to induce systemic necrosis. Systemic necrosis both in tomato and Nicotiana benthamiana shared hypersensitive response (HR) features, allowing the assessment of the role of different genomic regions on necrosis induction. Overexpression of both TGB3 and the polymerase domain (POL) of the RNA-dependent RNA polymerase (RdRp) resulted in necrosis, although only local expression of POL triggered HR-like symptoms. Our results also indicated that the necrosis-eliciting activity of POL resides in its highly conserved "palm" domain, and that necrosis was jasmonic acid-dependent but not salicylic acid-dependent. Altogether, our data suggest that the RdRp-POL domain plays an important role in PepMV necrosis induction, with necrosis development depending on the virus accumulation level, which can be modulated by the nature of TGB3, host genotype and environmental conditions.

Melon necrotic spot virus Replication Occurs in Association with Altered Mitochondria.

Melon necrotic spot virus (MNSV) (genus Carmovirus, family Tombusviridae) is a single-stranded, positive-sense RNA virus that has become an experimental model for the analysis of cell-to-cell virus movement and translation of uncapped viral RNAs, whereas little is known about its replication. Analysis of the cytopathology after MNSV infection showed the specific presence of modified organelles that resemble mitochondria. Immunolocalization of the glycine decarboxylase complex (GDC) P protein in these organelles confirmed their mitochondrial origin. In situ hybridization and immunolocalization experiments showed the specific localization of positive-sense viral RNA, capsid protein (CP), and double-stranded (ds)RNA in these organelles meaning that replication of the virus takes place in association with them. The three-dimensional reconstructions of the altered mitochondria showed the presence of large, interconnected, internal dilations which appeared to be linked to the outside cytoplasmic environment through pores and/or complex structures, and with lipid bodies. Transient expression of MNSV p29 revealed that its specific target is mitochondria. Our data document the extensive reorganization of host mitochondria induced by MNSV, which provides a protected environment to viral replication, and show that the MNSV p29 protein is the primary determinant of this effect in the host.

Small RNA-Seq to Characterize Viruses Responsible of Lettuce Big Vein Disease in Spain.

The emerging lettuce big-vein disease (LBVD) is causing losses in lettuce production ranging from 30 to 70% worldwide. Several studies have associated this disease with Mirafiori lettuce big-vein virus (MiLBVV) alone or in mixed infection with lettuce big-vein associated virus (LBVaV). We used Illumina small RNA sequencing (sRNA-seq) to identify viruses present in symptomatic lettuce plants from commercial fields in Southern Spain. Data analysis using the VirusDetect tool showed the consistent presence of MiLBVV and LBVaV in diseased plants. Populations of MiLBVV and LBVaV viral small RNAs (sRNAs) were characterized, showing features essentially similar to those of other viruses, with the peculiarity of an uneven asymmetric distribution of MiLBVV virus-derived small RNAs (vsRNAs) for the different polarities of genomic RNA4 vs. RNAs1 to 3. Sanger sequencing of coat protein genes was used to study MiLBVV and LBVaV phylogenetic relationships and population genetics. The Spanish MiLBVV population was composed of isolates from three well-differentiated lineages and reflected almost all of the diversity reported for the MiLBVV species, whereas the LBVaV population showed very little genetic differentiation at the regional scale but lineage differentiation at a global geographical scale. Universal primers were used to detect and quantify the accumulation of MiLBVV and LBVaV in field samples; both symptomatic and asymptomatic plants from affected fields carried equal viral loads, with LBVaV accumulating at higher levels than MiLBVV.

Oxidative stress induction by Prunus necrotic ringspot virus infection in apricot seeds.

Prunus necrotic ringspot rvirus (PNRSV) was able to invade the immature apricot seed including the embryo. The amount of virus was very high inside the embryo compared with that present in the cotyledons. PNRSV infection produced an oxidative stress in apricot seeds as indicated by the increase in lipid peroxidation, measured as thiobarbituric acid-reactive substances. This lipid peroxidation increase was parallelled with an imbalance in the seed antioxidant enzymes. A significant decrease in the ascorbate-GSH cycle enzymes as well as in peroxidase (POX) activity took place in infected seeds, suggesting a low capability to eliminate H2O2. No changes in superoxide dismutase (SOD) or catalase activity were observed. A significant decrease in polyphenoloxidase (PPO) activity was also observed. Native PAGE revealed the presence of three different SOD activity bands in apricot seeds: a Mn-containing SOD and two CuZn-containing SODs. Only an isozyme with catalase, glutathione reductase (GR) or PPO activity was detected in both healthy and infected apricot seeds. Regarding POX staining, three bands with POX activity were detected in native gels in both healthy and infected seeds. The gel results emphasise that the drop detected in POX, GR and PPO activities in PNRSV-infected apricot seeds by kinetic analyses was also evident from the results obtained by native PAGE. The oxidative stress and the imbalance in the antioxidant systems from PNRSV-infected apricot seeds resemble the hypersensitive response observed in some virus-host interactions. This defence mechanism would inactivate PNRSV during seed formation and/or the storage period or even during seed germination. Those results can explain the decrease in seed germination and the low transmission of PNRSV by seeds in apricot trees.

Prunus necrotic ringspot virus Early Invasion and Its Effects on Apricot Pollen Grain Performance.

ABSTRACT The route of infection and the pattern of distribution of Prunus necrotic ringspot virus (PNRSV) in apricot pollen were studied. PNRSV was detected both within and on the surface of infected pollen grains. The virus invaded pollen during its early developmental stages, being detected in pollen mother cells. It was distributed uniformly within the cytoplasm of uni- and bicellular pollen grains and infected the generative cell. In mature pollen grains, characterized by their triangular shape, the virus was located mainly at the apertures, suggesting that PNRSV distribution follows the same pattern as the cellular components required for pollen tube germination and cell wall tube synthesis. PNRSV also was localized inside pollen tubes, especially in the growth zone. In vitro experiments demonstrated that infection with PNRSV decreases the germination percentage of pollen grains by more than half and delays the growth of pollen tubes by approximately 24 h. However, although PNRSV infection affected apricot pollen grain performance during germination, the presence of the virus did not completely prevent fertilization, because the infected apricot pollen tubes, once germinated, were able to reach the apricot embryo sacs, which, in the climatic conditions of southeastern Spain, mature later than in other climates. Thus, infected pollen still could play an important role in the vertical transmission of PNRSV in apricot.

cmv1 is a gate for Cucumber mosaic virus transport from bundle sheath cells to phloem in melon.

Cucumber mosaic virus (CMV) has the broadest host range among plant viruses, causing enormous losses in agriculture. In melon, strains of subgroup II are unable to establish a systemic infection in the near-isogenic line SC12-1-99, which carries the recessive resistance gene cmv1 from the accession PI 161375, cultivar 'Songwhan Charmi'. Strains of subgroup I overcome cmv1 resistance in a manner dependent on the movement protein. We characterized the resistance conferred by cmv1 and established that CMV-LS (subgroup II) can move from cell to cell up to the veins in the inoculated leaf, but cannot enter the phloem. Immunogold labelling at transmission electron microscopy level showed that CMV-LS remains restricted to the bundle sheath (BS) cells in the resistant line, and does not invade vascular parenchyma or intermediary cells, whereas, in the susceptible line 'Piel de Sapo' (PS), the virus invades all vein cell types. These observations indicate that the resistant allele of cmv1 restricts systemic infection in a virus strain- and cell type-specific manner by acting as an important gatekeeper for virus progression from BS cells to phloem cells. Graft inoculation experiments showed that CMV-LS cannot move from the infected PS stock into the resistant cmv1 scion, thus suggesting an additional role for cmv1 related to CMV transport within or exit from the phloem. The characterization of this new form of recessive resistance, based on a restriction of virus systemic movement, opens up the possibility to design alternative approaches for breeding strategies in melon.

Vertical transmission of Prunus necrotic ringspot virus: hitch-hiking from gametes to seedling.

The aim of this work was to follow Prunus necrotic ringspot virus (PNRSV) infection in apricot reproductive tissues and transmission of the virus to the next generation. For this, an analysis of viral distribution in apricot reproductive organs was carried out at different developmental stages. PNRSV was detected in reproductive tissues during gametogenesis. The virus was always present in the nucellus and, in some cases, in the embryo sac. Studies within infected seeds at the embryo globular stage revealed that PNRSV infects all parts of the seed, including embryo, endosperm and testa. In the torpedo and bent cotyledon developmental stages, high concentrations of the virus were detected in the testa and endosperm. At seed maturity, PNRSV accumulated slightly more in the embryo than in the cotyledons. In situ hybridization showed the presence of PNRSV RNA in embryos obtained following hand-pollination of virus-free pistils with infected pollen. Interestingly, tissue-printing from fruits obtained from these pistils showed viral RNA in the periphery of the fruits, whereas crosses between infected pistils and infected pollen resulted in a total invasion of the fruits. Taken together, these results shed light on the vertical transmission of PNRSV from gametes to seedlings.