An alternative pathway to plant cold tolerance in the absence of vacuolar invertase activity

To cope with cold stress, plants have developed antioxidation strategies combined with osmoprotection by sugars. In potato (Solanum tuberosum) tubers, which are swollen stems, exposure to cold stress induces starch degradation and sucrose synthesis. Vacuolar acid invertase (VInv) activity is a significant part of the cold-induced sweetening (CIS) response, by rapidly cleaving sucrose into hexoses and increasing osmoprotection. To discover alternative plant tissue pathways for coping with cold stress, we produced VInv-knockout lines in two cultivars. Genome editing of VInv in 'Desiree' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4 degrees C, sugar analysis indicated that the knockout lines showed low levels of CIS and maintained low acid invertase activity in storage. Surprisingly, the tuber parenchyma of vinv lines exhibited significantly reduced lipid peroxidation and reduced H2O2 levels. Furthermore, whole plants of vinv lines exposed to cold stress without irrigation showed normal vigor, in contrast to WT plants, which wilted. Transcriptome analysis of vinv lines revealed upregulation of an osmoprotectant pathway and ethylene-related genes during cold temperature exposure. Accordingly, higher expression of antioxidant-related genes was detected after exposure to short and long cold storage. Sugar measurements showed an elevation of an alternative pathway in the absence of VInv activity, raising the raffinose pathway with increasing levels of myo-inositol content as a cold tolerance response

Pharmaceutical remediation using Algal Turf Scrubber (ATS)

Pharmaceuticals have become contaminants of emerging concern due to their continued detection and negative effects in aquatic environments. To prevent the release of pharmaceuticals from wastewater facilities, Algal Turf Scrubbers (ATS) have been proposed as a supplemental treatment step. ATS utilize the natural bioremediation capability of algal biofilm to clean re-circulating water. The low input requirements and valuable algal products that can be extracted from an ATS make it a cost-effective and versatile system in pollution remediation. In this lab-scale study, ATS removal efficiency of three common pharmaceuticals (ibuprofen, diclofenac, and sulfamethoxazole (SMX)) was quantified, and the effect of pharmaceutical exposure on the biofilm examined. In the course of a week, the ATS removed 97% of ibuprofen, 80% of SMX, and 79% of diclofenac concentrations. Additionally, total nitrogen and phosphorus were removed up to 29% and 72%, respectively. Changes in fluorescence, pigment production, and biomass productivity were insignificant, suggesting no stress response of the algae exposed to pharmaceuticals. Overall, ATS were effective in pharmaceutical removal without reduction in biofilm function. Further investigation should be done to determine bioremediation mechanisms, biofilm composition, and system efficiency on a larger scale with seasonal variability

Characterization of a Mitogen-Activated Protein Kinase Gene from Cucumber Required for Trichoderma-Conferred Plant Resistance

The fungal biocontrol agent Trichoderma asperellum has been recently shown to induce systemic resistance in plants through a mechanism that employs jasmonic acid and ethylene signal transduction pathways. Mitogen-activated protein kinase (MAPK) proteins have been implicated in the signal transduction of a wide variety of plant stress responses. Here we report the identification and characterization of a Trichoderma-induced MAPK (TIPK) gene function in cucumber (Cucumis sativus). Similar to its homologs, wound-induced protein kinase, MPK3, and MPK3a, TIPK is also induced by wounding. Normally, preinoculation of roots with Trichoderma activates plant defense mechanisms, which result in resistance to the leaf pathogen Pseudomonas syringae pv lachrymans. We used a unique attenuated virus vector, Zucchini yellow mosaic virus (ZYMV-AGII), to overexpress TIPK protein and antisense (AS) RNA. Plants overexpressing TIPK were more resistant to pathogenic bacterial attack than control plants, even in the absence of Trichoderma preinoculation. On the other hand, plants expressing TIPK-AS revealed increased sensitivity to pathogen attack. Moreover, Trichoderma preinoculation could not protect these AS plants against subsequent pathogen attack. We therefore demonstrate that Trichoderma exerts its protective effect on plants through activation of the TIPK gene, a MAPK that is involved in signal transduction pathways of defense responses

Analysis of the RNA-Dependent RNA Polymerase 1 (RDR1) Gene Family in Melon

RNA-dependent RNA polymerase 1 (RDR1) plays a crucial defense role against plant viruses by secondary amplification of viral double-stranded RNA in the gene-silencing pathway. In this study, it was found that melon (Cucumis melo) encodes four RDR1 genes (CmRDR1a, b, c1 and c2) similar to the CsRDR1 gene family of cucumber (C. sativus). However, in contrast to cucumber, melon harbors a truncated CmRDR1b gene. In healthy plants, CmRDR1a was expressed, whereas the expression of CmRDR1c1/c2 was not detected. CmRDR1a expression level increased 20-fold upon cucumber mosaic virus (CMV) infection and was not increased in melon plants infected with zucchini yellow mosaic virus (ZYMV), cucumber vein yellowing virus (CVYV) and cucumber green mottle mosaic virus (CGMMV). The expression of CmRDR1c1/c2 genes was induced differentially by infection with viruses from different families: high levels of ~340-, 172- and 115-fold increases were induced by CMV, CVYV and CGMMV, respectively, and relatively low-level increases by potyvirus infection (4- to 6-fold). CMV mutants lacking the viral silencing suppressor 2b protein did not cause increased CmRDR1c/c2 expression; knockout of CmRDR1c1/c2 by CRISPR/Cas9 increased susceptibility to CMV but not to ZYMV. Therefore, it is suggested that the sensitivity of melon to viruses from different families is a result of the loss of function of CmRDR1b

Cucumber RDR1s and cucumber mosaic virus suppressor protein 2b association directs host defence in cucumber plants

RNA-dependent RNA polymerases (RDRs) regulate important aspects of plant development and resistance to pathogens. The role of RDRs in virus resistance has been demonstrated using siRNA signal amplification and through the methylation of viral genomes. Cucumber (Cucumis sativus) has four RDR1 genes that are differentially induced during virus infection: CsRDR1a, CsRDR1b, and duplicated CsRDR1c1/c2. The mode of action of CsRDR1s during viral infection is unknown. Transient expression of the cucumber mosaic virus (CMV)-2b protein (the viral suppressor of RNA silencing) in cucumber protoplasts induced the expression of CsRDR1c, but not of CsRDR1a/1b. Results from the yeast two-hybrid system showed that CsRDR1 proteins interacted with CMV-2b and this was confirmed by bimolecular fluorescence complementation assays. In protoplasts, CsRDR1s localized in the cytoplasm as punctate spots. Colocalization experiments revealed that CsRDR1s and CMV-2b were uniformly dispersed throughout the cytoplasm, suggesting that CsRDR1s are redistributed as a result of interactions. Transient overexpression of individual CsRDR1a/1b genes in protoplasts reduced CMV accumulation, indicating their antiviral role. However, overexpression of CsRDR1c in protoplasts resulted in relatively higher accumulation of CMV and CMVΔ2b. In single cells, CsRDR1c enhances viral replication, leading to CMV accumulation and blocking secondary siRNA amplification of CsRDR1c by CMV-2b protein. This suggests that CMV-2b acts as both a transcription factor that induces CsRDR1c (controlling virus accumulation) and a suppressor of CsRDR1c activity

Plant Disease Symptomatology: Cucumber Green Mottle Mosaic Virus (CGMMV)-Infected Cucumber Plants Exposed to Fluctuating Extreme Temperatures

Greenhouse-grown cucumber plants inspected during and following extreme variations in environmental temperatures showed new characteristics of cucumber green mottle mosaic virus (CGMMV) disease manifestations. An increasing occurrence of CGMMV disease recovery has been associated with a new phenotype, identified at early stages of a reemerging disease. Symptoms of bright yellow islands (BYIs), conspicuous amid a dark green surrounding tissue (DGS), were detected in up to 10% of symptomatic plants in net-houses showing 50–60% recovery following an extreme temperature wave. Importantly, similar CGMMV disease initiation stages were observed in infected cucumber plants exposed to low temperatures of ~16 °C, under conditions of both controlled growth chambers and a net-house exposed to environmental temperature fluctuations. Apparently, a wide range of fluctuating temperatures evoked gradual manifestations of a reemerging disease

A Distinct Arabidopsis Latent Virus 1 Isolate Was Found in Wild <i>Brassica hirta</i> Plants and Bees, Suggesting the Potential Involvement of Pollinators in Virus Spread

During our search for aphid-pathogenic viruses, a comovirus was isolated from wild asymptomatic Brassica hirta (white mustard) plants harboring a dense population of Brevicoryne brassicae aphids. The transmission-electron-microscopy visualization of purified virions revealed icosahedral particles. The virus was mechanically transmitted to plants belonging to Brassicaceae, Solanaceae, Amaranthaceae, and Fabaceae families, showing unique ringspot symptoms only on B. rapa var. perviridis plants. The complete viral genome, comprised of two RNA segments, was sequenced. RNA1 and RNA2 contained 5921 and 3457 nucleotides, respectively, excluding the 3′ terminal poly-adenylated tails. RNA1 and RNA2 each had one open-reading frame encoding a polyprotein of 1850 and 1050 amino acids, respectively. The deduced amino acids at the Pro-Pol region, delineated between a conserved CG motif of 3C-like proteinase and a GDD motif of RNA-dependent RNA polymerase, shared a 96.5% and 90% identity with the newly identified Apis mellifera-associated comovirus and Arabidopsis latent virus 1 (ArLV1), respectively. Because ArLV1 was identified early in 2018, the B. hirta comovirus was designated as ArLV1-IL-Bh. A high-throughput-sequencing-analyses of the extracted RNA from managed honeybees and three abundant wild bee genera, mining bees, long-horned bees, and masked bees, sampled while co-foraging in a Mediterranean ecosystem, allowed the assembly of ArLV1-IL-Bh, suggesting pollinators’ involvement in comovirus spread in weeds

Enhanced Host-Parasite Resistance Based on Down-Regulation of Phelipanche aegyptiaca Target Genes Is Likely by Mobile Small RNA

RNA silencing refers to diverse mechanisms that control gene expression at transcriptional and post-transcriptional levels which can also be used in parasitic pathogens of plants that Broomrapes (Orobanche/Phelipanche spp.) are holoparasitic plants that subsist on the roots of a variety of agricultural crops and cause severe negative effects on the yield and yield quality of those crops. Effective methods for controlling parasitic weeds are scarce, with only a few known cases of genetic resistance. In the current study, we suggest an improved strategy for the control of parasitic weeds based on trans-specific gene-silencing of three parasite genes at once. We used two strategies to express dsRNA containing selected sequences of three Phelipanche aegyptiaca genes PaACS, PaM6PR, and PaPrx1 (pma): transient expression using Tobacco rattle virus (TRV:pma) as a virus-induced gene-silencing vector and stable expression in transgenic tomato Solanum lycopersicum (Mill.) plants harboring a hairpin construct (pBINPLUS35:pma). siRNA-mediated transgene-silencing (20–24 nt) was detected in the host plants. Our results demonstrate that the quantities of PaACS and PaM6PR transcripts from P. aegyptiaca tubercles grown on transgenic tomato or on TRV-infected Nicotiana benthamiana plants were significantly reduced. However, only partial reductions in the quantity of PaPrx1 transcripts were observed in the parasite tubercles grown on tomato and on N. benthamiana plants. Concomitant with the suppression of the target genes, there were significant decreases in the number and weight of the parasite tubercles that grew on the host plants, in both the transient and the stable experimental systems. The results of the work carried out using both strategies point to the movement of mobile exogenous siRNA from the host to the parasite, leading to the impaired expression of essential parasite target genes