Sensitive and Visual Detection of Brassica Yellows Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification-Coupled CRISPR-Cas12 Assay.

Brassica yellows virus (BrYV) is an economically important virus on cruciferous species. In this study, a one-pot reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was developed for the detection of BrYV. The limit of detection of this method reached 32.8 copies of the BrYV ORF5, which is 100-fold more sensitive than the RT-LAMP method. Moreover, there was no cross-reactivity with other rapeseed-infecting RNA viruses or poleroviruses. We dried the CRISPR/Cas12a reagent in a trehalose and pullulan mixture to retain its efficacy at the RT-LAMP temperature of 63°C in order to allow portable BrYV detection in a water bath. The entire process can be performed in about 1 h, and a positive result can be rapidly and conveniently detected using a handheld UV lamp. In the field, the RT-LAMP-CRISPR/Cas12a assay was accurate and had higher sensitivity than RT-LAMP and reverse transcription-polymerase chain reaction assays. The novel RT-LAMP-CRISPR/Cas12a assay allows convenient, portable, rapid, low-cost, highly sensitive, and specific detection of BrYV and has great potential for on-site monitoring of BrYV.

Two novel transporters NtNRAMP6a and NtNRAMP6b are involved in cadmium transport in tobacco (Nicotiana tabacum L.).

Plant natural resistance-associated macrophage protein (NRAMP) plays important roles in metal transport and tolerance. Tobacco is a typical cadmium (Cd) accumulator, while research on NRAMP in tobacco has been limited. In the current study, two novel NRAMP genes (NtNRAMP6a and NtNRAMP6b) were identified from the allotetraploid plant Nicotiana tabacum L. Real time‒PCR and GUS (ß-glucuronidase) staining results showed that the two genes were expressed in roots, stems, leaves and flowers and induced by Cd stress. Subcellular localization revealed that they were located in the plasma membrane. Heterologously expressed NtNRAMP6a and NtNRAMP6b significantly increased the Cd sensitivity of the Δycf1 mutant, indicating that NtNRAMP6a and NtNRAMP6b had Cd transport functions in yeast. The difference in the manganese (Mn) transport activity of the two genes was demonstrated by point mutation, which was caused by the difference in the 18th amino acid. NRAMP6-N18K is a new key active site for manganese transport. After 50 µM Cd treatment for 7 days, the contents of Cd and Mn of the ntnramp6a/6b mutants was significantly lower than those of wild type in shoots, while the contents in roots were higher. Additionally, the mutant lines showed higher chorphyll contentration and lighter leaf damage. Knockout of NtNRAMP6a and NtNRAMP6b reduced Cd and Mn accumulation in tobacco shoots by influence root-to-shoot translocation. This provides new idea for cultivating tobacco varieties with low cadmium accumulation and high cadmium tolerance.

The OPEN STOMATA1-SPIRAL1 module regulates microtubule stability during abscisic acid-induced stomatal closure in Arabidopsis.

Drought stress triggers abscisic acid (ABA) signaling in guard cells and induces stomatal closure to prevent water loss in land plants. Stomatal movement is accompanied by reorganization of the cytoskeleton. Cortical microtubules disassemble in response to ABA, which is required for stomatal closure. However, how ABA signaling regulates microtubule disassembly is unclear, and the microtubule-associated proteins (MAPs) involved in this process remain to be identified. In this study, we show that OPEN STOMATA 1 (OST1), a central component in ABA signaling, mediates microtubule disassembly during ABA-induced stomatal closure in Arabidopsis thaliana. We identified the MAP SPIRAL1 (SPR1) as the substrate of OST1. OST1 interacts with and phosphorylates SPR1 at Ser6, which promotes the disassociation of SPR1 from microtubules and facilitates microtubule disassembly. Compared with the wild type, the spr1 mutant exhibited significantly greater water loss and reduced ABA responses, including stomatal closure and microtubule disassembly in guard cells. These phenotypes were restored by introducing the phosphorylated active form of SPR1. Our findings demonstrate that SPR1 positively regulates microtubule disassembly during ABA-induced stomatal closure, which depends on OST1-mediated phosphorylation. These findings reveal a specific connection between a core component of ABA signaling and MAPs.

Cloning, characterization and functional analysis of NtMYB306a gene reveals its role in wax alkane biosynthesis of tobacco trichomes and stress tolerance.

Trichomes are specialized hair-like organs found on epidermal cells of many terrestrial plants, which protect plant from excessive transpiration and numerous abiotic and biotic stresses. However, the genetic basis and underlying mechanisms are largely unknown in Nicotiana tabacum (common tobacco), an established model system for genetic engineering and plant breeding. In present study, we identified, cloned and characterized an unknown function transcription factor NtMYB306a from tobacco cultivar K326 trichomes. Results obtained from sequence phylogenetic tree analysis showed that NtMYB306a-encoded protein belonged to S1 subgroup of the plants' R2R3-MYB transcription factors (TFs). Observation of the green fluorescent signals from NtMYB306a-GFP fusion protein construct exhibited that NtMYB306a was localized in nucleus. In yeast transactivation assays, the transformed yeast containing pGBKT7-NtMYB306a construct was able to grow on SD/-Trp-Ade+X-α-gal selection media, signifying that NtMYB306a exhibits transcriptional activation activity. Results from qRT-PCR, in-situ hybridization and GUS staining of transgenic tobacco plants revealed that NtMYB306a is primarily expressed in tobacco trichomes, especially tall glandular trichomes (TGTs) and short glandular trichomes (SGTs). RNA sequencing (RNA-seq) and qRT-PCR analysis of the NtMYB306a-overexpressing transgenic tobacco line revealed that NtMYB306a activated the expression of a set of key target genes which were associated with wax alkane biosynthesis. Gas Chromatography-Mass Spectrometry (GC-MS) exhibited that the total alkane contents and the contents of n-C28, n-C29, n-C31, and ai-C31 alkanes in leaf exudates of NtMYB306a-OE lines (OE-3, OE-13, and OE-20) were significantly greater when compared to WT. Besides, the promoter region of NtMYB306a contained numerous stress-responsive cis-acting elements, and their differential expression towards salicylic acid and cold stress treatments reflected their roles in signal transduction and cold-stress tolerance. Together, these results suggest that NtMYB306a is necessarily a positive regulator of alkane metabolism in tobacco trichomes that does not affect the number and morphology of tobacco trichomes, and that it can be used as a candidate gene for improving stress resistance and the quality of tobacco.

Molecular Characterization of a Novel Polerovirus Infecting Soybean in China.

Poleroviruses are positive-sense, single-stranded viruses. In this study, we describe the identification of a novel polerovirus isolated from soybean displaying curled leaves. The complete viral genome sequence was identified using high-throughput sequencing and confirmed using rapid amplification of cDNA ends (RACE), RT-PCR and Sanger sequencing. Its genome organization is typical of the members of genus Polerovirus, containing seven putative open reading frames (ORFs). The full genome is composed of single-stranded RNA of 5822 nucleotides in length, with the highest nucleotide sequence identity (79.07% with 63% coverage) for cowpea polerovirus 2 (CPPV2). Amino acid sequence identities of the protein products between the virus and its relatives are below the threshold determined by the International Committee of Taxonomy of Viruses (ICTV) for species demarcation, and this strongly supports this virus' status as a novel species, for which the name soybean chlorotic leafroll virus (SbCLRV) is proposed. Recombination analysis identified a recombination event in the ORF5 of the 3' portion in the genome. Phylogenetic analyses of the genome and encoded protein sequences revealed that the new virus is closely related to phasey bean mild yellows virus, CPPV2 and siratro latent polerovirus. Subsequently, we demonstrated the infectivity of SbCLRV in Nicotiana benthamiana via infectious cDNA clone generation and agroinoculation.

Anti-TMV activity and effects of three prieurianin-type limonoids from Munronia henryi.

Three prieurianin-type limonoids (1-3), including two new compounds (1 and 2) and one known compound (3) were isolated from Munronia henryi. These compounds were tested for their activities against tobacco mosaic virus (TMV) by the conventional half-leaf method and the outcomes were analyzed by western blotting and RT-PCR assays. The three tested compounds, at 100 µg/mL, showed strong antiviral activities in the pretreated tobacco plants with inhibition rates ranging from 70.5% to 81.3%, which were significantly higher than that of the positive control, ningnanmycin (55.6%). Their potential of inducing systemic acquired resistance (SAR) was also evaluated, in which compound 1 showed excellent induction activities. Furthermore, it was found that potentiation of defense-related enzyme activity and the contents of SA was increased. Compound 1 could also inhibit the expression of TMV CP and up-regulate the expression of defense-related genes. This work revealed that these limonoids, especially compound 1 could induce resistance in tobacco plants against the viral pathogen TMV. Meanwhile, compounds 1-3 could down-regulate the expression of NtHsp70-1 and Nthsp70-261 genes, indicating that these limonoids possibly inhibit TMV infection by suppressing NtHsp70-1 and Nthsp70-261 expression. This study is the first to report antiviral compounds with two different mechanisms of action.

Triplex Immunostrip Assay for Rapid Diagnosis of Tobacco Mosaic Virus, Tobacco Vein Banding Mosaic Virus, and Potato Virus Y.

Mixed virus infection has increasingly become a problem in the production of Solanaceae crops in recent years; therefore, a fast and accurate detection method is needed. In this study, a novel triplex immunostrip assay was developed for the simultaneous detection of tobacco mosaic virus (TMV), tobacco vein banding mosaic virus (TVBMV), and potato virus Y (PVY). The limits of detection of this novel immunostrip reached 200 ppb (ng/ml), 1 ppm (µg/ml), and 2 ppm for TMV, PVY, and TVBMV particles, respectively. Importantly, no cross-reactivity was observed among TMV, TVBMV, and PVY or to a nontarget virus. When the assay was applied to suspected virus-infected tobacco, tomato, and potato samples collected from fields in Southwest China, samples of single or mixed virus infection were successfully identified. In conclusion, the triplex immunostrip assay provides a fast and easy to use on-site detection method for field epidemiological studies of TMV, TVBMV, and PVY, and for managing diseases that are caused by them.

Haploid induction in allotetraploid tobacco using DMPs mutation.

MAIN CONCLUSION: dmp1dmp2dmp3 mutants created by CRISPR/Cas9 could trigger maternal haploids in the allotetraploid model plant Nicotiana tabacum L. Double haploid (DH) technology is becoming increasingly important because it can significantly accelerate the breeding process. Haploid induction plays a fundamental role in the production of DH lines. Haploid induction has been realized and applied in diploid plants using DMP genes. However, it has yet to be elucidated whether haploid induction could be established in polyploid plants. In the current study, three homologues of the DMP genes (NtDMP1, 2, and 3) were identified in the allotetraploid plant Nicotiana tabacum, and the encoded proteins localized in the endoplasmic reticulum. Loss-of-function mutations in all three genes triggered maternal haploids with an induction rate of 1.52-1.75%. Compared with wild-type tobacco, the created haploid inducer exhibited differences in pollen vigor and seed germination rate. Furthermore, to rapidly and easily screen haploids, a visible haploid identification system was established based on a powdery mildew resistance phenotype. Findings from this study lay the foundation for the potential application of haploid inducers in allotetraploid plants such as tobacco.

Geminiviruses boost active DNA demethylation for counter-defense.

DNA methylation regulates gene expression under abiotic and biotic stresses. Recently, Gui et al. discovered that geminiviruses subverted DNA methylation-mediated defense through boosting the active DNA demethylation mediated by host DNA glycosylases to promote viral virulence. Their findings reveal a distinctive counter-defense strategy exploited by invading pathogens to achieve successful infection.

Types of Interferons and Their Expression in Plant Systems.

Interferons (IFNs) are divided into 3 types (type I, type II, and type III) on the basis of sequence homology and functional properties. Recombinant IFNs have been approved by regulatory agencies in many countries for clinical treatment of hepatitis B, hepatitis C, and other diseases; these IFNs are mainly produced in microorganisms and mammalian cell systems. However, there are serious obstacles to the production of recombinant IFNs in microorganism systems; for example, the recombinant IFN may have different glycosylation patterns from the native protein, be present in insoluble inclusion bodies, be contaminated with impurities such as endotoxins and nucleic acids, have a short half-life in human blood, and incur high production costs. Some medicinal proteins have been successfully expressed in plants and used in clinical applications, suggesting that plants may also be a good system for IFN expression. However, there are still many technical problems that need to be addressed before the clinical application of plant-expressed IFNs, such as increasing the amount of recombinant protein expression and ensuring that the IFN is modified with the correct type of glycosylation. In this article, we review the classification of IFNs, their roles in antiviral signal transduction pathways, their clinical applications, and their expression in plant systems.

Identification and genome analysis of a tomato zonate spot virus isolate from Bidens pilosa.

Bidens pilosa is a weed species that invades crop areas in tropical and subtropical regions. To date, only two potyviruses have been reported to infect B. pilosa. Here, we report the complete genome sequence of a tomato zonate spot tospovirus (TZSV) isolate from Bidens named TZSV-Bidens. The tripartite RNA of the TZSV-Bidens genome contains L, M, and S segments that are 8912, 4724, and 2997 nt in length, respectively. The genome contains five open reading frames (ORFs), with 92.23-95.01% amino acid sequence identity to the TZSV-YN isolate. Phylogenetic analysis based on amino acid sequences of members of the family Tospoviridae showed that TZSV-Bidens was grouped into a well-supported Eurasian cluster. The intergenic regions (IGRs) of the M and S RNAs are among the most variable regions and are far shorter than those of the TZSV-YN reference genome.

Development and application of a full-length infectious clone of potato virus Y isolate belonging to SYR-I strain.

Potato virus Y (PVY) causes huge damage to potato and tobacco production worldwide. The complete genome sequence of GZ, a PVY isolate (strain SYR-I) from Guizhou province, China, was cloned into the binary vector pCambia0390. Three introns were individually inserted into the P3 and CI ORFs to produce plasmid pCamPVY-GZ. The plasmid could infect plants of Nicotiana benthamiana, N. tabacum via agroinfiltration and plants of pepper and potato by mechanical inoculation. The green fluorescence protein gene of Aequoria victoriae was cloned into the encoding regions between nuclear inclusion body 'b' and coat protein genes in pCamPVY-GZ to produce pCamPVY-GZ-GFP, which could infect plants of N. benthamiana, N. tabacum, potato and tomato, and produce green fluorescence in the systemic leaves of inoculated plants. Mutations were introduced to pCamPVY-GZ to make the lysine (K) 391 and glutamic acid (E)410 of helper component-proteinase to arginine (R) and asparagic acid (E), respectively. Unlike wild type PVY-GZ, the mutant PVY-K391R/E410D could not induce veinal necrosis in N. tabacum plants. With an interval of 14 days, mutant PVY-K391R/E410D could protect N. tabacum plants from the infection of severe PVY strain. The results presented here provide a promising alternate for the prevention of diseases caused by PVY.

Integrated analysis of tobacco miRNA and mRNA expression profiles under PVY infection provids insight into tobacco-PVY interactions.

Potato virus Y (PVY) is a globally and economically important pathogen of potato, tobacco, tomato and other staple crops and caused significant yield losses and reductions in quality.To explore the molecular PVY-host interactions, we analysed changes in the miRNA and mRNA profiles of tobacco in response to PVY infection. A total of 81 differentially expressed miRNAs belonging to 29 families and 8133 mRNAs were identified. The Gene Ontology (GO) enrichment analyses showed that genes encoding the DNA/RNA binding, catalytic activity and signalling molecules were all significantly enriched. Moreover, 88 miRNA-mRNA interaction pairs were identified through a combined analysis of the two datasets. We also found evidence showing that the virus-derived siRNAs (vsiRNAs) from the PVY genome target tobacco translationally controlled tumor protein (NtTCTP) mRNA and mediate plant resistance to PVY. Together, our findings revealed that both miRNA and mRNA expression patterns can be changed in response to PVY infection and novel vsiRNA-plant interactions that may regulate plant resistance to PVY. Both provide fresh insights into the virus-plant interactions.

Molecular variation and recombination in RNA segment 10 of rice black-streaked dwarf virus isolated from China during 2007-2010.

Rice black-streaked dwarf virus (RBSDV) is the causal agent of rice black-streaked dwarf and maize rough dwarf diseases in China. The only open reading frame encoding the viral outer capsid protein on S10 RNA of 21 RBSDV isolates was sequenced for phylogenetic and recombination analysis. Four natural recombinants were detected, and the recombinant breakpoints were identified. In addition, the distribution of synonymous and non-synonymous nucleotide changes revealed that the virus had been evolving under purifying selection.

Alteration of gene expression profile in maize infected with a double-stranded RNA fijivirus associated with symptom development.

Maize rough dwarf disease caused by Rice black-streaked dwarf virus (RBSDV) is a major viral disease in China. It has been suggested that the viral infection of plants might cause distinct disease symptoms through the inhibition or activation of host gene transcription. We scanned the gene expression profile of RBSDV-infected maize through oligomer-based microarrays to reveal possible expression changes associated with symptom development. Our results demonstrate that various resistance-related maize genes and cell wall- and development-related genes, such as those for cellulose synthesis, are among the genes whose expression is dramatically altered. These results could aid in research into new strategies to protect cereal crops against viruses, and reveal the molecular mechanisms of development of specific symptoms in rough dwarf-related diseases.