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 'Désirée' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4°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 H2 O2 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.

Development of Powdery Mildew Resistance in Cucumber Using CRISPR/Cas9-Mediated Mutagenesis of CsaMLO8.

Powdery mildew (PM) diseases may severely limit the production of various crops, including members of the family Cucurbitaceae. Successful PM infection relies on the Mildew Resistance Locus O (MLO) plant gene family, which encodes susceptibility factors essential for fungus penetration into the host cell. In cucumber (Cucumis sativus), natural mutations in CsaMLO8 confer resistance to the PM pathogen Podosphaera xanthii. Here, we used CRISPR/Cas9-mediated mutagenesis to generate PM resistance in the susceptible cucumber cultivar Ilan. Two transgene-free Csamlo8 CRISPR mutant lines (Csamlo-cr-1 and Csamlo-cr-2) were isolated, the first with a 5-bp deletion in exon 1, and the second harboring a 1,280-bp deletion and 10-bp insertion between exons 1 and 5. Both lines showed high resistance to PM under semicommercial growth conditions in the summer growing seasons of 2019 and 2021. These results provide the basis for generating transgene-free powdery mildew resistance in cucumber in any genetic background. This method can directly be employed on commercial cultivars and hybrid parental lines, and thereby substantially shorten and simplify the breeding process for PM resistance in cucumber.

Sucrose promotes stem branching through cytokinin.

Shoot branching is an important aspect of plant architecture because it substantially affects plant biology and agricultural performance. Sugars play an important role in the induction of shoot branching in several species, including potato (Solanum tuberosum L.). However, the mechanism by which sugars affect shoot branching remains mostly unknown. In the present study, we addressed this question using sugar-mediated induction of bud outgrowth in potato stems under etiolated conditions. Our results indicate that sucrose feeding to detached stems promotes the accumulation of cytokinin (CK), as well as the expression of vacuolar invertase (VInv), an enzyme that contributes to sugar sink strength. These effects of sucrose were suppressed by CK synthesis and perception inhibitors, while CK supplied to detached stems induced bud outgrowth and VInv activity in the absence of sucrose. CK-induced bud outgrowth was suppressed in vinv mutants, which we generated by genome editing. Altogether, our results identify a branching-promoting module, and suggest that sugar-induced lateral bud outgrowth is in part promoted by the induction of CK-mediated VInv activity.

Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA.

Gene silencing is a natural defense response of plants against invading RNA and DNA viruses. The RNA post-transcriptional silencing system has been commonly utilized to generate transgenic crop plants that are "immune" to plant virus infection. Here, we applied this approach against the devastating DNA virus tomato yellow leaf curl virus (TYLCV) in its host tomato (Solanum lycopersicum L.). To generate broad resistance to a number of different TYLCV viruses, three conserved sequences (the intergenic region [NCR], V1-V2 and C1-C2 genes) from the genome of the severe virus (TYLCV) were synthesized as a single insert and cloned into a hairpin configuration in a binary vector, which was used to transform TYLCV-susceptible tomato plants. Eight of 28 independent transgenic tomato lines exhibited immunity to TYLCV-Is and to TYLCV-Mld, but not to tomato yellow leaf curl Sardinia virus, which shares relatively low sequence homology with the transgene. In addition, a marker-free (nptII-deleted) transgenic tomato line was generated for the first time by Agrobacterium-mediated transformation without antibiotic selection, followed by screening of 1180 regenerated shoots by whitefly-mediated TYLCV inoculation. Resistant lines showed a high level of transgene-siRNA (t-siRNA) accumulation (22% of total small RNA) with dominant sizes of 21 nt (73%) and 22 nt (22%). The t-siRNA displayed hot-spot distribution ("peaks") along the transgene, with different distribution patterns than the viral-siRNA peaks observed in TYLCV-infected tomato. A grafting experiment demonstrated the mobility of 0.04% of the t-siRNA from transgenic rootstock to non-transformed scion, even though scion resistance against TYLCV was not achieved.

A Distinct Arabidopsis Latent Virus 1 Isolate Was Found in Wild Brassica hirta 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.

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.

Knockout of SlTOM1 and SlTOM3 results in differential resistance to tobamovirus in tomato.

During tobamovirus-host coevolution, tobamoviruses developed numerous interactions with host susceptibility factors and exploited these interactions for replication and movement. The plant-encoded TOBAMOVIRUS MULTIPLICATION (TOM) susceptibility proteins interact with the tobamovirus replicase proteins and allow the formation of the viral replication complex. Here CRISPR/Cas9-mediated mutagenesis allowed the exploration of the roles of SlTOM1a, SlTOM1b, and SlTOM3 in systemic tobamovirus infection of tomato. Knockouts of both SlTOM1a and SlTOM3 in sltom1a/sltom3 plants resulted in an asymptomatic response to the infection with recently emerged tomato brown rugose fruit virus (ToBRFV). In addition, an accumulation of ToBRFV RNA and coat protein (CP) in sltom1a/sltom3 mutant plants was 516- and 25-fold lower, respectively, than in wild-type (WT) plants at 12 days postinoculation. In marked contrast, sltom1a/sltom3 plants were susceptible to previously known tomato viruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), indicating that SlTOM1a and SlTOM3 are not essential for systemic infection of TMV and ToMV in tomato plants. Knockout of SlTOM1b alone did not contribute to ToBRFV and ToMV resistance. However, in triple mutants sltom1a/sltom3/sltom1b, ToMV accumulation was three-fold lower than in WT plants, with no reduction in symptoms. These results indicate that SlTOM1a and SlTOM3 are essential for the replication of ToBRFV, but not for ToMV and TMV, which are associated with additional susceptibility proteins. Additionally, we showed that SlTOM1a and SlTOM3 positively regulate the tobamovirus susceptibility gene SlARL8a3. Moreover, we found that the SlTOM family is involved in the regulation of plant development.

A high level of transgenic viral small RNA is associated with broad potyvirus resistance in cucurbits.

Gene-silencing has been used to develop resistance against many plant viruses but little is known about the transgenic small-interfering RNA (t-siRNA) that confers this resistance. Transgenic cucumber and melon lines harboring a hairpin construct of the Zucchini yellow mosaic potyvirus (ZYMV) HC-Pro gene accumulated different levels of t-siRNA (6 to 44% of total siRNA) and exhibited resistance to systemic ZYMV infection. Resistance to Watermelon mosaic potyvirus and Papaya ring spot potyvirus-W was also observed in a cucumber line that accumulated high levels of t-siRNA (44% of total siRNA) and displayed significantly increased levels of RNA-dependent RNA (RDR)1 and Argonaute 1, as compared with the other transgenic and nontransformed plants. The majority of the t-siRNA sequences were 21 to 22 nucleotides in length and sense strand biased. The t-siRNA were not uniformly distributed throughout the transgene but concentrated in "hot spots" in a pattern resembling that of the viral siRNA peaks observed in ZYMV-infected cucumber and melon. Mutations in ZYMV at the loci associated with the siRNA peaks did not break this resistance, indicating that hot spot t-siRNA may not be essential for resistance. This study shows that resistance based on gene-silencing can be effective against related viruses and is probably correlated with t-siRNA accumulation and increased expression of RDR1.

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.

New early phenotypic markers for cucumber green mottle mosaic virus disease in cucumbers exposed to fluctuating extreme temperatures.

Studies of early stages of cucumber green mottle mosaic virus (CGMMV) disease have been recently focused on plant molecular responses. However, extreme diurnal environmental temperatures, characteristic of global climate changes, could affect plant susceptibility and disease phenotype progression. Our studies of CGMMV disease progression, under simulated extreme temperature waves, have revealed two new disease initiation phenotypes that developed gradually, preceding severe symptom manifestations of post-recovery CGMMV systemic infections. 'Early post-recovery stage' bright yellow islands (BYIs) with defined boundaries amid asymptomatic leaf blades were first emerging followed by 'late post-recovery stage' BYIs with diffused boundaries. A deduced CGMMV disease progression scheme, postulating BYI symptom occurrence time-windows, revealed BYIs in field grown cucumber plants exposed to extreme diurnal temperatures. Profiling ontology of cucumber differentially expressed genes in BYIs vs the associated dark-green surrounding tissues disclosed activation of jasmonic acid (JA) pathway in 'early post-recovery stage' BYIs. JA signaling was inactivated in 'late post-recovery stage' BYIs concomitant with increasing expressions of JA signaling inhibitors and downregulation of JA responsive phenylpropanoid pathway. Our results disclosed a new phenotypic description of CGMMV disease initiation, characteristic of cucumbers grown under extreme environmental temperature fluctuations. The BYI phenotypes could define a time-window for CGMMV disease management applications.

Tomato plants transformed with the inhibitor-of-virus-replication gene are partially resistant to Botrytis cinerea.

Tomato plants transformed with a cDNA clone encoding the inhibitor-of-virus-replication (IVR) gene were partially resistant to Botrytis cinerea. This resistance was observed as a significant reduction in the size of lesions induced by the fungus in transgenic plants compared with the lesions on the nontransgenic control plants. This resistance was weakened when plants were kept at an elevated temperature, 32 degrees C, before inoculation with B. cinerea compared with plants kept at 17 to 22 degrees C prior to inoculation. Resistance correlated with the presence of IVR transcripts, as detected by reverse transcription-polymerase chain reaction. This is one of the few cases in which a gene associated with resistance to a virus also seems to be involved in resistance to a fungal disease.

Tomato Brown Rugose Fruit Virus Contributes to Enhanced Pepino Mosaic Virus Titers in Tomato Plants.

The tobamovirus tomato brown rugose fruit virus (ToBRFV), a major threat to tomato production worldwide, has recently been documented in mixed infections with the potexvirus pepino mosaic virus (PepMV) CH2 strain in traded tomatoes in Israel. A study of greenhouse tomato plants in Israel revealed severe new viral disease symptoms including open unripe fruits and yellow patched leaves. PepMV was only detected in mixed infections with ToBRFV in all 104 tested sites, using serological and molecular analyses. Six PepMV isolates were identified, all had predicted amino acids characteristic of CH2 mild strains excluding an isoleucine at amino acid position 995 of the replicase. High-throughput sequencing of viral RNA extracted from four selected symptomatic plants showed solely the ToBRFV and PepMV, with total aligned read ratios of 40.61% and 11.73%, respectively, indicating prevalence of the viruses. Analyses of interactions between the co-infecting viruses by sequential and mixed viral inoculations of tomato plants, at various temperatures, showed a prominent increase in PepMV titers in ToBRFV pre-inoculated plants and in mixed-infected plants at 18-25 °C, compared to PepMV-single inoculations, as analyzed by Western blot and quantitative RT-PCR tests. These results suggest that Israeli mild PepMV isolate infections, preceded by ToBRFV, could induce symptoms characteristic of PepMV aggressive strains.

Gene silencing of mannose 6-phosphate reductase in the parasitic weed Orobanche aegyptiaca through the production of homologous dsRNA sequences in the host plant.

Orobanche spp. (broomrape) are parasitic plants which subsist on the roots of a wide range of hosts, including tomato, causing severe losses in yield quality and quantity. Large amounts of mannitol accumulate in this parasitic weed during development. Mannose 6-phosphate reductase (M6PR) is a key enzyme in mannitol biosynthesis, and it has been suggested that mannitol accumulation may be very important for Orobanche development. Therefore, the Orobanche M6PR gene is a potential target for efforts to control this parasite. Transgenic tomato plants were produced bearing a gene construct containing a specific 277-bp fragment from Orobanche aegyptiaca M6PR-mRNA, in an inverted-repeat configuration. M6PR-siRNA was detected in three independent transgenic tomato lines in the R1 generation, but was not detected in the parasite. Quantitative RT-PCR analysis showed that the amount of endogenous M6PR mRNA in the tubercles and underground shoots of O. aegyptiaca grown on transgenic host plants was reduced by 60%-80%. Concomitant with M6PR mRNA suppression, there was a significant decrease in mannitol level and a significant increase in the percentage of dead O. aegyptiaca tubercles on the transgenic host plants. The detection of mir390, which is involved with cytoplasmic dsRNA processing, is the first indication of the existence of gene-silencing mechanisms in Orobanche spp. Gene silencing mechanisms are probably involved with the production of decreased levels of M6PR mRNA in the parasites grown on the transformed tomato lines.

Broomrape can acquire viruses from its hosts.

Broomrapes (Phelipanche, formerly Orobanche) are parasitic plants that physically connect with the vascular systems of their hosts through haustorial structures. In this study, we found that Cucumber mosaic virus (CMV), Tomato mosaic virus (ToMV), Potato virus Y (PVY), and Tomato yellow leaf curl virus (TYLCV) translocate from infected host plants to Phelipanche aegyptiaca. In order to examine whether these viruses, and specifically CMV, replicate in the parasite, we tested several replication parameters. We detected accumulation of both plus and minus strands of CMV genomic RNA and CMV-derived siRNAs in the shoots of Phelipanche grown on CMV-infected tobacco and tomato plants. We purified CMV particles from Phelipanche grown on CMV-infected plants. These particles were present in amounts comparable to those found in the hosts' leaves. These data indicate that CMV replicates in Phelipanche tissues. In addition, viable ToMV and PVY were observed, and the plus and minus strand RNAs of ToMV were detected in Phelipanche shoots grown on infected hosts. However, we found only low levels of ToMV coat protein and did not detect any PVY coat protein. We also detected genomic TYLCV DNA in shoots of Phelipanche grown on TYLCV-infected tomato. Thus, for the first time, we demonstrate that broomrape is a host for at least one plant virus CMV, and possibly various other viruses.

The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development.

The helper component-proteinase (HC-Pro) protein of potyviruses is a suppressor of gene silencing and has been shown to elicit plant developmental-defect-like symptoms. In Zucchini yellow mosaic virus (ZYMV), a mutation in the highly conserved FR180NK box of HC-Pro to FI180NK causes attenuation of these symptoms. At 5 days postinoculation and before symptoms appear, virus accumulation, HC-Pro protein levels, and viral short interfering RNA (siRNA) levels are similar for the severe (FRNK) and attenuated (FINK) strains. At this stage, ZYMV(FRNK) caused greater accumulation of most microRNAs (miRNAs), and especially of their complementary miRNA "passenger" strands (miRNA*s), in systemically infected leaves than the attenuated ZYMV(FINK) did. HC-Pro(FRNK) specifically bound artificial siRNA and miRNA/miRNA* duplexes with a much higher affinity than the mutated HC-Pro(FINK). Further analysis of the mutant and wild-type HC-Pro proteins revealed that suppressor activity of the ZYMV HC(FINK) mutant was not diminished. However, the FINK mutation caused a loss of HC-Pro suppressor function in other potyviruses. Replacement of the second positively charged amino acid in the ZYMV FRNK box to result in FRNA also caused symptom attenuation and reduced small RNA duplex-binding affinity without loss of suppressor activity. Our data suggest that the highly conserved FRNK box in the HC-Pro of potyviruses is a probable point of contact with siRNA and miRNA duplexes. The interaction of the FRNK box with populations of miRNAs directly influences their accumulation levels and regulatory functions, resulting in symptom development.

Differential expression of cucumber RNA-dependent RNA polymerase 1 genes during antiviral defence and resistance.

RNA-dependent RNA polymerase 1 (RDR1) plays a crucial role in plant defence against viruses. In this study, it was observed that cucumber, Cucumis sativus, uniquely encodes a small gene family of four RDR1 genes. The cucumber RDR1 genes (CsRDR1a, CsRDR1b and duplicated CsRDR1c1/c2) shared 55%-60% homology in their encoded amino acid sequences. In healthy cucumber plants, RDR1a and RDR1b transcripts were expressed at higher levels than transcripts of RDR1c1/c2, which were barely detectable. The expression of all four CsRDR1 genes was induced by virus infection, after which the expression level of CsRDR1b increased 10-20-fold in several virus-resistant cucumber cultivars and in a broad virus-resistant transgenic cucumber line expressing a high level of transgene small RNAs, all without alteration in salicylic acid (SA) levels. By comparison, CsRDR1c1/c2 genes were highly induced (25-1300-fold) in susceptible cucumber cultivars infected with RNA or DNA viruses. Inhibition of RDR1c1/c2 expression led to increased virus accumulation. Ectopic application of SA induced the expression of cucumber RDR1a, RDR1b and RDRc1/c2 genes. A constitutive high level of RDR1b gene expression independent of SA was found to be associated with broad virus resistance. These findings show that multiple RDR1 genes are involved in virus resistance in cucumber and are regulated in a coordinated fashion with different expression profiles.

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.

Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology.

Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N' and C' termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off-target sites. Non-transgenic heterozygous eif4e mutant plants were selected for the production of non-transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. In contrast, heterozygous mutant and non-mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non-transgenically, not visibly affecting plant development and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.

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.