Plant Cell Reports

Ferns and their allies (monilophytes) represent the second most species-rich group of land plants and are of considerable ecological importance. As a sister group of seed plants (including flowering plants) they are also of great evolutionary interest. Compared to flowering plants, however, much less is known about the developmental and molecular biology of ferns. Among the most important reasons have been the huge genome sizes of ferns and technical obstacles such as the lack of an efficient transformation system. In recent years the situation has improved considerably, however. For the fern model system Ceratopteris richardii a whole genome sequence has been published, and an efficient transformation system has been developed. To further facilitate studies on fern biology we aim at simplifying genome engineering of C. richardii with the CRISPR-Cas9 system. We report C. richardii plants that express Cas9 nuclease under control of the strong CaMV 35S promoter. For efficient expression of single guide RNA (sgRNA) by RNA polymerase III we identified C. richardii U6 promoters. These technical improvements may foster many fields of fern physiology, development and evolution.

We studied the biochemical properties of three splicing isoforms of PR-10 from rubber tree (Hevea brasiliensis) and found that purified recombinant HbPR10 can cause RNA degradation in vitro, a well-known activity described for many PR-10 proteins. This ribonuclease activity was observed for all three HbPR10 splicing isoforms and is abolished by boiling. However, inclusion of a negative control proteins revealed that ribonuclease activity rather originates from RNases that are copurified from E. coli, which are overlooked by traditionally used controls such as heat inactivation, RNase inhibitors and negative control proteins obtained with different procedures. The crucial control proteins are missing for at least nine reports on ribonuclease activity in PR-10 proteins published by different laboratories worldwide, indicating that proper controls are frequently overlooked in ribonuclease assays. The raised cautionary note applies to several PR-10 proteins with proclaimed ribonuclease activities and call for the use of different assays and mutant PR-10 proteins as control.

Biological invasions remain a major global challenge. Alligatorweed (Alternanthera philoxeroides (Mart.) Griseb.), native to South America, has had profound negative effects on ecosystem function and economy in Australia, North America, and Asia. It is an invasive and primarily aquatic plant that, despite a documented lack of genetic diversity, is unusually adaptive - thriving in both aquatic and terrestrial environments. However, genetic resources for studying this invasive plant are limited. Here, we have assembled the transcriptome of alligatorweed using all publicly available cDNA sequences. The resulting assembly produced over 500K contigs with an average length of [~]700 bases and an N50 of >1000 bases and contains over 100K probable gene-coding sequences. Although this assembly is slightly smaller than the previously published assembly developed from just cold-treated shoot tips, the new assembly is slightly more complete with over 95% of the conserved plant genes being represented as full length transcripts, and only 2.3% of these conserved genes being unrepresented compared to 2.7% missing in the earlier assembly. Resources from the PANTHER database were used to annotate all transcripts containing long open reading frames. Comparisons to several plant species identified gene ontologies that were over- and under-represented in the alligatorweed transcriptome including cellular transport and cytoskeletal processes and cell signaling, which could explain the high growth rate and phenotypic plasticity that make alligatorweed particularly invasive. We also sequenced and assembled a genomic database for alligatorweed using only short read technologies. This assembly produced over ten million contigs with an average length of only 300 bases and an N50 of 451 bases. However, 88% of the transcripts were represented among the genomic contigs, indicating that these contigs could serve as a source for regulatory elements for genes previously shown to be differentially expressed under various conditions. Kmer analysis indicated that 22% of the alligatorweed genome was comprised of repetitive elements. A similarity search against the plant repetitive element database indicated that long terminal repeat containing elements including copia- and gypsy-like elements made up the bulk of the transposons present in the alligatorweed genome. Additionally, we assembled and annotated a full-length chloroplast and a partial mitochondrial genome. Combined, these resources provide a source of gene sequences that should be useful for more complete genomic assemblies and for investigating gene structure and function in this particularly adaptable and invasive species. The results will provide an excellent starting point for many different investigations into the biology and ecology of alligatorweed, strengthen our understanding of the invasiveness, biology and ecology of invasive plants, and will help develop a reasonable management strategy to reduce risk and costs of the impacts.

Translation is a fundamental process for every living organism. In plants, the rate of translation is tightly modulated during development and in response to environmental cues. However, it is difficult to measure the actual translation state of the tissues in vivo. Here, we report the implementation of an in vivo translation marker based on bimolecular fluorescence complementation, the Ribo-BiFC. We combined method originally developed for fruit-fly with an improved low background split-mVenus BiFC system previously described in plants. We labelled Arabidopsis thaliana small subunit ribosomal protein (RPS) and large subunit ribosomal protein (RPL) with fragments of the mVenus fluorescent protein. Upon the assembly of the 80S ribosome, the mVenus fragments complemented and were detected by fluorescent microscopy. We show that these recombinant proteins are in close proximity in the tobacco epidermal cells, although the signal is reduced when compared to BiFC signal from known interactors. This Ribo-BiFC method system can be used in stable transgenic lines to enable visualisation of translational rate in plant tissues and could be used to study translation dynamics and its changes during plant development, under abiotic stress or in different genetic backgrounds.

During Agrobacterium-mediated transformation (AMT), T-DNA along with several virulence proteins like VirD2, VirE2, VirE3, VirD5, and VirF enter into the plant cytoplasm. VirE2 is supposed to serve as single-stranded DNA binding (SSB) protein and assist the cytoplasmic trafficking of T-DNA inside the host cell. In the present study, a rice glutathione-S-transferase (OsGSTU5) that interacts with VirE2 protein in plant cytoplasm has been identified. OsGSTU5 is observed to be involved in post-translational glutathionylation of VirE2 protein (gVirE2). In silico analysis revealed that gVirE2+ssDNA complex is structurally less stable than VirE2+ ssDNA complex. The gel shift activity confirms the attenuated SSB property of gVirE2 over VirE2 protein under in vitro condition. Moreover, knock-down and overexpression OsGSTU5 phenotypes of rice showed increased and decreased T-DNA expression, respectively after Agrobacterium infection. The present finding convincingly establishes the role of OsGSTU5 as defense protein in rice that can further serve as an important target for modulation of AMT efficiency in rice.

In 2020 we suffered from a major global pandemic caused by the SARS-CoV-2 coronavirus. Efforts to contain the virus include the development of rapid tests and vaccines, which require a ready supply of viral proteins. Here we report the production of two SARS-CoV-2 proteins by transient transformation of tobacco, leading to high expression within three days, and subsequent purification of the intact proteins. Such efforts may help to develop testing resources to alleviate the major impacts of this global pandemic.

Recent studies have shown that the primary transcripts of some microRNA genes (pri-miRNAs) are able to express short proteins (peptides) ranging usually from 12-15 amino acid residues to around 30 residues in length. These peptides, called miPEPs, may participate in the regulation of transcription of their own pri-miRNAs. Using bioinformatic comparative analysis of pri-miRNA sequences in plant genomes, we previously discovered a new group of miPEPs (miPEP-156a), which is encoded by pri-miR156a in several dozen species from the Brassicaceae family. Exogenous peptides miPEP-156a can effectively penetrate plant seedlings through the root system and spread systemically to the leaves of young seedlings. At the same time, a moderate morphological effect is observed, which consists in accelerated growth of the main root of the seedling. In parallel, a positive effect is observed at the level of pri-miR156a expression. It is important that the effects at the morphological and molecular levels are seemingly related to the ability of the peptide to quickly transfer into the cell nuclei and bind to nuclear chromatin. In this work, the secondary structure of the peptide was also experimentally established, and changes in this structure in the complex with DNA were shown.

Beet curly top Iran Virus (BCTIV) is a yield-limiting geminivirus belonging to the becurtovirus genus. The genome organization of BCTIV is unique such that the complementary strand of BCTIV resembles mastreviruses, whereas the virion strand organization is close to curtoviruses. Geminiviruses are known to avoid the plant defense system by suppressing the RNA interference mechanisms both at the transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) level. Multiple geminivirus genes have been identified as viral suppressors of RNA silencing (VSR) but VSR activity remains elusive in becurtoviruses. By screening all verified open reading frames in the BCTIV genome, we found that only V2 and Rep were able to suppress specific PTGS mechanisms, triggered by the expression of a partial or full-length sense-strand transcript of the target gene (S-PTGS). BCTIV-V2 could suppress S-PTGS more efficiently than BCTIV-Rep when then the target GFP gene is transiently expressed. On the other hand, S-PTGS is suppressed by Rep but not V2 when target GFP is only stably expressed. Deletional mutagenesis of BCTIV-Rep implicated that multiple domains are required for its VSR activity. Furthermore, neither V2 nor Rep could fully suppress local PTGS induced by inverted repeat targeting GFP (GFP-IR). Also, in a closer look at the spread of local silencing by GFP-IR, we observed that V2 or Rep are not able to suppress the movement of sRNAs. Nevertheless, Rep suppressed the systemic silencing induced by GFP-IR in 16C plants. Northern blot analyses showed that BCTIV-Rep inhibits silencing by mitigating sRNA production, whereas BCTIV-V2 does not alter sRNA levels. In summary, both the silencing phenotype and the molecular signatures of silencing implicate distinct modes of VSR activity of BCTIV-Rep and -V2.

Passion fruit (Passiflora edulis) is a perennial, woody, tropical vine crop. It produces edible round to oval fruit that has been increasingly favored for its unique aroma and taste, and richness in antioxidants, vitamins and minerals. However, the functional genomic study of passion fruit lags far behind due to a lack of simple and efficient genetic tools. Here, we report the development of virus-mediated protein overexpression (VOX) and virus-induced gene silencing (VIGS) vector based on telosma mosaic virus (TelMV), an emerging potyvirus infecting passion fruit plants worldwide. This vector, designated pTelMV-GW, incorporates the Gateway-compatible recombination sites for rapid gene cloning. We show that this vector allows for the systemic stable expression of two heterologous proteins, green fluorescent protein (GFP) and bacterial phytoene synthase (crtB) in passion fruit plants, and pTelMV-GW containing different fragments of GFP can also induce systemic gene silencing on the GFP-transgenic N. benthamiana plants. Moreover, we demonstrated that in passion fruit plants, this vector can trigger gene silencing of endogenous phytoene desaturase (PDS) to a limited extent. Furthermore, we upgraded the vector by using a mild TelMV strain that does not induce noticeable symptoms in plants. We show that the upgraded vector (pTelMV-R181K-GW) containing PDS or ChlI fragments induces the robust silencing of the corresponding endogenous gene in passion fruit plants. Together, we reported the first development of VIGS and VOX vectors in passion fruit plants, as the first step in our endeavor to discover horticulturally important genes for improving passion fruit production and quality.

Papaya (Carica papaya L.) is an economically important tropical crop that produces papain and highly nutritious fruit, which are used in the grocery, cosmetic, pharmaceutical, and food processing industries. However, various destructive pathogens severely threaten its production. Furthermore, limited natural genetic variation restricts breeding efforts for crop improvement. Therefore, we turned to gene editing as a tool to address these problems. We utilized two CRISPR systems (Cas9 and Cas12a) and two papaya genes, CpPDS (phytoene desaturase) and CpMLO6 (Mildew Locus O 6), to establish efficient genome editing systems of papaya. The systems were delivered by an optimized protocol of Agrobacterium-mediated transformation (AMT) of embryogenic callus suspension cultures derived from hypocotyls. Accordingly, we transformed papaya with five plasmid constructs, each of which expressed one or two guide RNAs (gRNAs) for gene editing using either Cas9 or Cas12a. All except two T0 transgenic plants tested produced mutations with the majority containing indels of over 90%. Furthermore, successful mutation of the CpPDS gene using both Cas9 and Cas12a produced albino phenotypes as expected for disrupting a gene for carotenoid biosynthesis. Successful mutagenesis was achieved with seven out of eight gRNAs. Homozygous and/or biallelic mutants were generated from transformation using all five constructs, suggesting the feasibility of obtaining transgene-free homozygous segregating mutants by selfing in the second generation. Taken together, a robust and reliable papaya genome editing system was established, which enables genetic modification in various genomic environments to meet the diverse needs of basic scientific research and tropical crop improvement.

Passion fruit (Passiflora edulis) is grown perennially in sub-tropical and tropical areas. Its fruits contain multiple vitamins and antioxidants, and thus are consumed increasingly in drinks and foods. However, the functions and regulations of genes that are engaged in the biosynthesis of the health-promoting compounds in passion fruits remain largely unknown. Its whole genome sequence has just been published recently. Virus-induced gene silencing (VIGS) is a reverse genetics tool for analyzing gene function. Here, we engineered telosma mosaic virus (TelMV), a potyvirus infecting passion fruit, into a VIGS vector by inserting the Gateway-compatible recombination sites. The newly constructed TelMV-VIGS virus successfully expressed foreign protein and induced systemic infection in both Nicotiana benthamiana and P. edulis plants. Intriguingly, TelMV-VIGS vector containing different fragments of green fluorescent protein (GFP) gene induced systemic gene silencing on the GFP-transgenic N. benthamiana plants (16c). When the phytoene desaturase (PDS) gene, an endogenous gene in passion fruit, was engineered into the vector, it triggered the silencing of PePDS, as evidenced by the reduced mRNA levels and photobleached phenotype. We reported the first development of VIGS vector in passion fruit, as the first step in our endeavor of discovering horticulturally important genes for improving passion fruit production and quality.

Nucleotide-binding Leucine Rich repeat-type immune receptors (NLRs) are intracellular proteins that sense the presence of pathogen-derived elicitors and subsequently trigger an immune response. NLR proteins have to be strictly regulated as immune responses typically result in death of affected host cells. Regulation mechanisms of NLR activation are well studied, however steps immediately following NLR activation are largely unexplained. Multimerization of NLRs is thought to be involved, although currently no unambiguous paradigm regarding the dynamics of this process exists. Some NLRs form high-molecular weight complexes before activation, others exclusively after activation, or, like Rx1, none could be detected. We investigated NLR complex formation in transgenic N. benthamiana stably expressing the potato Rx1 protein from its native promoter. Activation of the Rx1-resistance response was synchronized by dexamethasone-controlled expression of its elicitor; the Potato Virus X Coat Protein. Rx1 self-associates upon activation: Rx1 homomers are absent before dexamethasone application, a complex could be detected 1 hour after application, but surprisingly is again absent after 2 hours or later. These results show that self-association of NLR proteins upon activation can be transient, explaining the difficulties of detecting them during the normal, non-synchronized infection process as this typically involves few affected cells at any time.

Chloroplast perturbations activate retrograde signalling pathways causing dynamic changes of gene expression. Besides transcriptional control of gene expression different classes of small non-coding RNAs (sRNAs) act in gene expression control, but comprehensive analyses regarding their role in retrograde signalling is lacking. We performed sRNA profiling in response to norflurazon (NF) that provokes retrograde signals in A. thaliana wild type and the two retrograde signalling mutants gun1 and gun5. The RNA samples were also used for mRNA and long non-coding RNA (lncRNA) profiling to link altered sRNA levels to changes of their cognate target RNAs. We identified 122 sRNAs from all known sRNA classes that were responsive to NF in wild type. Strikingly, 140 and 213 sRNAs were found to be differentially regulated in both mutants indicating a retrograde control of these sRNAs. Concomitant with the changes in sRNA expression we detected about 1500 differentially expressed mRNAs in the NF treated wild type and around 900 and 1400 mRNAs that were differentially regulated in the gun1 and gun5 mutant with a high proportion (~30%) of genes encoding plastid proteins. Furthermore, around 20% of predicted miRNA targets code for plastid localised proteins. The analyses of sRNA-target pairs identified pairs with an anticorrelated expression as well pairs showing other expressional relations pointing to a role of sRNAs in balancing transcriptional changes upon retrograde signals. Based on the comprehensive changes in sRNA expression we assume a considerable impact of sRNAs in retrograde-dependent transcriptional changes to adjust plastidic and nuclear gene expression. Significance statementPerturbations of plastid functions trigger retrograde signalling to adjust plastidic and nuclear gene expression, however, the role of small non-coding RNAs acting as regulators in these pathways is not well understood. We analysed small non-coding RNA expression in response to retrograde signals in A. thaliana wild type and two retrograde signalling mutants and identified members of all known small non-coding RNA classes pointing to a functional role of these RNA classes in retrograde pathways.

Gametophyte-specific promoters drive the expression of genes in male and/or female gametophytes. These have applications in breeding experiments, gene function identification, developmental biology-related studies, and off-lately in genome editing also. The Plant Intracellular Ras-group Leucine-Rich-Repeat 6 (PIRL6) gene is known to be necessary for male and female gametogenesis. Using the GUS- based deletion analysis, we have identified the PIRL6 promoter length that is essential for exclusive expression in the male gametophyte of Arabidopsis thaliana. We studied the strength of various lengths of PIRL6 promoters in different tissues, by GUS expression quantification. The male-gametophyte-specific promoter segment (PA1) exhibited stronger expression in mature anthers than the younger ones. We identified 50 other genes that co-expressed with PIRL6 in Arabidopsis using the Expression Angler tool. Gene ontology (GO) analysis shows that these 51 co-expressing genes were predominantly involved in cell differentiation. By comparing the promoter sequences of these 51 genes, the presence of three over-represented known motifs, POLLEN1LELAT52, ACGTATERD1 and CIACADIANLELHC was identified. We could also predict the presence of three novel cis-regulatory elements in the co-expressing gene network using the MEME suite tool. Additionally, we confirmed the functionality of the ABRE and P-box elements present in PIRL6 promoter using the tobacco leaf transient assay. Thus, we cloned and functionally confirmed the promoter region of PIRL6 required for male gametophyte-specific expression, compared this promoter with those of 50 other co-expressed genes and predicted their functions, and analyzed their cis regulatory regions and predicted three novel motifs also. Key messageA pollen-specific promoter fragment of PIRL6 was functionally characterized using GUS-based deletion analysis. Fifty other co-expressed genes were compared and novel cis-regulatory elements were predicted. Two hormone-responsive elements in the PIRL6 promoter were found to influence the GUS expression. PA1 promoter can be used in experiments that require male gametophyte-specific expression of genes.

Functional validation of genetic components in plants often requires cloning them separately into both plant and bacterial expression vectors, a process that is both time-consuming and laborious. This study aimed to simplify this workflow by developing plant-bacteria dual-host promoter systems that drive high-level constitutive expression in both environments. To achieve this, two variants of the chloramphenicol acetyltransferase promoter (PCAT), a bacterial {sigma} factor-dependent promoter, were integrated into the cauliflower mosaic virus 35S promoter (P35S), and their performance was evaluated using a hygromycin phosphotransferase (HPT)-GFP fusion reporter. One of these variants, PCAT1, conferred hygromycin resistance to Escherichia coli (DH5 and BL21 (DE3)) and maintained high-level expression comparable to the original P35S in onion epidermal cells. A hybrid P35S enhancer-PNOS system also conferred hygromycin resistance to E. coli, but its activity in inducing GFP signals in onion cells remained lower than that of P35S. Due to its compact size (89 bp) and efficiency, PCAT1 can serve as a module for converting standard plant vectors into dual-host systems, accelerating gene characterization and the development of new gene-based tools.

Eggplant (Solanum melongena L.) is one of the most important vegetables grown and consumed in the Philippines; hence, continuous breeding programs are vital to maintain the supply of this economically important crop. This study demonstrates the first successful Agrobacterium-mediated genetic transformation and plant regeneration of a Philippine eggplant cultivar, PH 11424 also known as Mistisa. Cotyledons from two-week old seedlings were used as explants, which were transformed with disarmed Agrobacterium tumefaciens strain LBA4404 harboring a binary vector for CRISPR/Cas9 expression and hygromycin phosphotransferase (HPT), an antibiotic selection marker. Growth of shoot primordia from the agro-infected explants was observed during selective culture with 7.5 ppm hygromycin, which indicated an initial transformation success. The putatively transformed shoot primordia were then transferred to an elongation medium. The elongated and hygromycin-resistant shoots were allowed to develop roots in hormone-free medium supplemented with hygromycin, and subsequently acclimatized under greenhouse conditions. The entire process took at least 5 - 6 months. Of the total 585 agro-infected explants, the regeneration efficiency of rooted shoots was 5.1%. Successful transformation was confirmed by polymerase chain reaction (PCR) amplification of Cas9. Acclimatized plants tested positive for the transgene. The transgenic eggplants successfully reached maturity, flowered, and set seed. These results demonstrate a working Agrobacterium-mediated transformation and plant regeneration protocols using cotyledons as explants in a Philippine eggplant genotype. These biotechnology tools are critical for the successful application of genetic engineering (GE) and new breeding techniques (NBTs) in eggplant crop improvement.

Plant extracellular vesicles (EVs) have become the focus of rising interest due to their important roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes to modulate pathogen virulence. However, the isolation of pure intact EVs from plants still represents a considerable challenge. Currently, plant EVs have been isolated from apoplastic washing fluid (AWF) using a variety of methods. Here, we compare two published methods used for isolating plant EVs, and provide a detailed recommended method for AWF collection from Arabidopsis thaliana, followed by EV isolation via differential ultracentrifugation. To further separate and purify specific subclasses of EV from heterogeneous vesicles, sucrose or iodixanol density-based separation and immunoaffinity capture are then utilized. We found that immunoaffinity capture provides a significant advantage for specific EV isolation when suitable specific EV biomarkers and their corresponding antibodies are available. Overall, this study guides the selection and optimization of EV isolation methods for desired downstream applications.

Sheath Blight (SB) disease in rice crop caused by the infection of the fungal pathogen Rhizoctonia solani (R. solani) is one of the severe rice diseases that can cause up to 50% yield losses. Naturally occurring rice varieties resistant to SB have not been reported yet. We have performed a Time-Series RNA-Seq analysis on a widely cultivated rice variety BPT-5204 for identifying its transcriptomic response signatures to R. solani infection at 1st, 2nd and 5th day post inoculation (dpi). In total, 428, 3225 and 1225 genes were differentially expressed in the treated rice plants post 1, 2 and 5 dpi, respectively. GO and KEGG enrichment analysis identified significant processes and pathways differentially altered in the rice plant after the fungal infection. Machine learning and network based integrative approach was used to construct Transcriptional Regulatory Networks (TRNs) of the rice plant at the three Time Points. Regulatory network analysis identified SUB1B, MYB30 and CCA1 as important regulatory hub Transcription Factors in rice during R. solani infection. Jasmonic acid signaling pathway was activated and in contrast, photosynthesis and carbon fixation processes were significantly compromised. Involvement of MAPK, CYPs, Peroxidases and PAL genes was observed in response to the fungal infection. Circadian clock was also strongly influenced by R. solani infection. Our integrative analysis identified 7 putative SB resistant genes altered in rice after R. solani infection and provided a better understanding of rice plant response to R. solani infection. One sentence summaryTime series expression analysis of rice variety BPT-5204 identifies key molecular signatures involved in rice plant response to R. solani infection.

Fagus sylvatica L. (European beech) is a dominant hardwood forest tree species across Central Europe, supporting diverse ecosystem services and forming the basis of a significant market for high-value timber. However, climate change increasingly threatens beech vitality and productivity, making molecular insights into its stress resilience and functional validation of underlying genes urgently needed. Here, we report an improved protocol for protoplast isolation from seedling leaves and demonstrate, for the first time, transient genetic transformation and CRISPR/Cas-mediated genome editing in F. sylvatica. PEG-mediated transformation was sequentially optimized, achieving efficiencies of 59 {+/-} 6.19%. Transformation efficiency was strongly influenced by season, which also affected protoplast yield. Both, a basic molecular toolkit for functional genomics and future biotechnological applications were established by testing a set of promoters and reporters. For proof-of-concept genome editing, we achieved 4.75 to 32.69% editing efficiencies in the PHYTOENE DESATURASE gene (FsPDS) using temperature-tolerant LbCas12a (ttLbCas12a). The establishment of a reliable protoplast transformation and editing system provides a crucial foundation for future genetic improvement and functional studies in this non-model tree species.

SummarySystems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as the Cas nucleases or the guide RNA (gRNA). While the Cas nucleases are constant elements in editing approaches, gRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of gRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing (VIGE). In this work, we engineered Potato virus X (PVX) to build a vector able to easily express one or more gRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expressed Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced gRNAs achieving highly efficient multiplex editing in a few days in adult plant tissues. We also demonstrate that genome modifications are inherited in plants regenerated from infected tissues. In sum, the new PVX VIGE vector allows easy, fast and efficient expression of gRNAs arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in important crops of the family Solanaceae.Competing Interest StatementThe authors have declared no competing interest.View Full Text