Heritable gene editing in tomato through viral delivery of isopentenyl transferase and single-guide RNAs to latent axillary meristematic cells.

Realizing the full potential of genome editing for crop improvement has been slow due to inefficient methods for reagent delivery and the reliance on tissue culture for creating gene-edited plants. RNA viral vectors offer an alternative approach for delivering gene engineering reagents and bypassing the tissue culture requirement. Viruses, however, are often excluded from the shoot apical meristem, making virus-mediated gene editing inefficient in some species. Here, we developed effective approaches for generating gene-edited shoots in Cas9-expressing transgenic tomato plants using RNA virus-mediated delivery of single-guide RNAs (sgRNAs). RNA viral vectors expressing sgRNAs were either delivered to leaves or sites near axillary meristems. Trimming of the apical and axillary meristems induced new shoots to form from edited somatic cells. To further encourage the induction of shoots, we used RNA viral vectors to deliver sgRNAs along with the cytokinin biosynthesis gene, isopentenyl transferase. Abundant, phenotypically normal, gene-edited shoots were induced per infected plant with single and multiplexed gene edits fixed in the germline. The use of viruses to deliver both gene editing reagents and developmental regulators overcomes the bottleneck in applying virus-induced gene editing to dicotyledonous crops such as tomato and reduces the dependency on tissue culture.

Differential Tropism in Roots and Shoots of Resistant and Susceptible Cassava (Manihot esculenta Crantz) Infected by Cassava Brown Streak Viruses.

Cassava brown streak disease (CBSD) is a destructive disease of cassava in Eastern and Central Africa. Because there was no source of resistance in African varieties to provide complete protection against the viruses causing the disease, we searched in South American germplasm and identified cassava lines that did not become infected with the cassava brown streak viruses. These findings motivated further investigations into the mechanism of virus resistance. We used RNAscope® in situ hybridization to localize cassava brown streak virus in cassava germplasm lines that were highly resistant (DSC 167, immune) or that restricted virus infections to stems and roots only (DSC 260). We show that the resistance in those lines is not a restriction of long-distance movement but due to preventing virus unloading from the phloem into parenchyma cells for replication, thus restricting the virus to the phloem cells only. When DSC 167 and DSC 260 were compared for virus invasion, only a low CBSV signal was found in phloem tissue of DSC 167, indicating that there is no replication in this host, while the presence of intense hybridization signals in the phloem of DSC 260 provided evidence for virus replication in companion cells. In neither of the two lines studied was there evidence of virus replication outside the phloem tissues. Thus, we conclude that in resistant cassava lines, CBSV is confined to the phloem tissues only, in which virus replication can still take place or is arrested.

High efficient de novo root-to-shoot organogenesis in Citrus jambhiri Lush.: Gene expression, genetic stability and virus indexing.

A protocol for high-frequency direct organogenesis from root explants of Kachai lemon (Citrus jambhiri Lush.) was developed. Full-length roots (~3 cm) were isolated from the in vitro grown seedlings and cultured on Murashige and Skoog basal medium supplemented with Nitsch vitamin (MSN) with different concentrations of cytokinin [6-benzylaminopurine, (BAP)] and gibberellic acid (GA3). The frequency of multiple shoot proliferation was very high, with an average of 34.3 shoots per root explant when inoculated on the MSN medium supplemented with BAP (1.0 mg L-1) and GA3 (1.0 mg L-1). Optimal rooting was induced in the plantlets under half strength MSN medium supplemented with indole-3-acetic acid (IAA, 0.5-1.0 mg L-1). IAA induced better root structure than 1-naphthaleneacetic acid (NAA), which was evident from the scanning electron microscopy (SEM). The expressions of growth regulating factor genes (GRF1 and GRF5) and GA3 signaling genes (GA2OX1 and KO1) were elevated in the regenerants obtained from MSN+BAP (1.0 mg L-1)+GA3 (1.0 mg L-1). The expressions of auxin regulating genes were high in roots obtained in ½ MSN+IAA 1.0 mg L-1. Furthermore, indexing of the regenerants confirmed that there was no amplicons detected for Huanglongbing bacterium and Citrus tristeza virus. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers detected no polymorphic bands amongst the regenerated plants. This is the first report that describes direct organogenesis from the root explant of Citrus jambhiri Lush. The high-frequency direct regeneration protocol in the present study provides an enormous significance in Citrus organogenesis, its commercial cultivation and genetic conservation.

The Potential of Cryotherapy to Remove Sugarcane Mosaic Virus from Sugarcane (Saccharum spp. hybrids) Shoot Tips.

BACKGROUND: Virus-free sugarcane is difficult to achieve due to the multiple vegetative propagation cycles employed commercially. In vitro culture using small (1 mm) meristematic shoot tips has eliminated viruses but survival is low with small explants. OBJECTIVE: Droplet-Vitrification (D-V) and V-Cryoplate protocols were investigated for the elimination of Sugarcane mosaic virus (SCMV) from large (c. 3 mm) in vitro-derived shoot tips. MATERIALS AND METHODS: Shoot tips excised from NCo376 and N19 cultivars were exposed to both cryogenic procedures. Virus indexing by RT-qPCR was performed 16 weeks after recovery. RESULTS: Explants exposed to cryo-treatments that recovered and multiplied was 30-92%, while at least 90% of control explants regenerated. No virus was detected in multiplied shoots from either cultivar after D-V and liquid nitrogen immersion. In NCo376, virus was eliminated after D-V without cooling. CONCLUSION: The preliminary findings suggest that cryotherapy and/or osmotherapy are viable options for SCMV removal from infected plants.

A New Perspective on Cryotherapy: Pathogen Elimination Using Plant Shoot Apical Meristem via Cryogenic Techniques.

Plant pathogens cause different diseases on crops and industrial plant species that result in economic losses. Pathogen-free plant material has usually been obtained by traditional procedures such as meristem culture, thermotherapy, and chemotherapy. However, there are many limitations of these procedures such as mechanical challenges of meristem excision and low regeneration rate, low resistance to high temperatures, phytotoxicity, and mutagenic effects of the chemicals used in the procedures. Cryotherapy is a newly developed biotechnological tool that has been very effective in virus elimination from economically important plant species. This tool has overcome the abovementioned limitations. This chapter aims to highlight the importance of the cryogenic procedures (vitrification, encapsulation-vitrification, droplet vitrification, two-step freezing, dehydration, encapsulation-dehydration) in order to generate virus-free germplasm.

Lettuce Chlorosis Virus Disease: A New Threat to Cannabis Production.

In a survey conducted in Cannabis sativa L. (cannabis) authorized farms in Israel, plants showed disease symptoms characteristic of nutrition deprivation. Interveinal chlorosis, brittleness, and occasional necrosis were observed in older leaves. Next generation sequencing analysis of RNA extracted from symptomatic leaves revealed the presence of lettuce chlorosis virus (LCV), a crinivirus that belongs to the Closteroviridae family. The complete viral genome sequence was obtained using RT-PCR and Rapid Amplification of cDNA Ends (RACE) PCR followed by Sanger sequencing. The two LCV RNA genome segments shared 85-99% nucleotide sequence identity with LCV isolates from GenBank database. The whitefly Bemisiatabaci Middle Eastern Asia Minor1 (MEAM1) biotype transmitted the disease from symptomatic cannabis plants to un-infected 'healthy' cannabis, Lactucasativa, and Catharanthusroseus plants. Shoots from symptomatic cannabis plants, used for plant propagation, constituted a primary inoculum of the disease. To the best of our knowledge, this is the first report of cannabis plant disease caused by LCV.

Arbuscular Mycorrhizal Symbiosis Affects Plant Immunity to Viral Infection and Accumulation.

Arbuscular mycorrhizal (AM) fungi, as root symbionts of most terrestrial plants, improve plant growth and fitness. In addition to the improved plant nutritional status, the physiological changes that trigger metabolic changes in the root via AM fungi can also increase the host ability to overcome biotic and abiotic stresses. Plant viruses are one of the important limiting factors for the commercial cultivation of various crops. The effect of AM fungi on viral infection is variable, and considerable attention is focused on shoot virus infection. This review provides an overview of the potential of AM fungi as bioprotection agents against viral diseases and emphasizes the complex nature of plant-fungus-virus interactions. Several mechanisms, including modulated plant tolerance, manipulation of induced systemic resistance (ISR), and altered vector pressure are involved in such interactions. We propose that using "omics" tools will provide detailed insights into the complex mechanisms underlying mycorrhizal-mediated plant immunity.

Cryobiotechnology: A Double-Edged Sword for Obligate Plant Pathogens.

Pathogen-free stock plants are required as propagation materials in nurseries and healthy materials are needed in germplasm exchange between countries or regions through quarantine programs. In addition, plant gene banks also prefer to maintain pathogen-free germplasm collections. Shoot tip cryotherapy is a novel biotechnology method whereby cryopreservation methods are used to eradicate obligate pathogens from vegetatively propagated plants. Long-term preservation of pathogens is necessary in all types of virus-related basic research and applications such as antigen preparation for virus detection by immunology-based methods, production of plant-based vaccines, genetic transformation to produce virus-derived resistant transgenic plants, and bionanotechnology to produce nano drugs. Obligate plant pathogens such as viruses and viroids are intracellular parasites that colonize only living cells of the hosts. Therefore, their long-term preservation is difficult. Cryotreatments cannot completely eradicate the obligate pathogens that do not infect meristematic cells and certain proportions of plants recovered from cryotreatments are still pathogen-infected. Furthermore, cryotreatments often fail to eradicate the obligate pathogens that infect meristematic cells. Cryopreservation can be used for the long-term cryopreservation of the obligate plant pathogens. Thus, cryobiotechnology functions as a double-edged sword for plant pathogen eradication and cryopreservation. This review provides updated a synthesis of advances in cryopreservation techniques for eradication and cryopreservation of obligate plant pathogens.

Protein composition of the occlusion bodies of Epinotia aporema granulovirus.

Within family Baculoviridae, members of the Betabaculovirus genus are employed as biocontrol agents against lepidopteran pests, either alone or in combination with selected members of the Alphabaculovirus genus. Epinotia aporema granulovirus (EpapGV) is a fast killing betabaculovirus that infects the bean shoot borer (E. aporema) and is a promising biopesticide. Because occlusion bodies (OBs) play a key role in baculovirus horizontal transmission, we investigated the composition of EpapGV OBs. Using mass spectrometry-based proteomics we could identify 56 proteins that are included in the OBs during the final stages of larval infection. Our data provides experimental validation of several annotated hypothetical coding sequences. Proteogenomic mapping against genomic sequence detected a previously unannotated ac110-like core gene and a putative translation fusion product of ORFs epap48 and epap49. Comparative studies of the proteomes available for the family Baculoviridae highlight the conservation of core gene products as parts of the occluded virion. Two proteins specific for betabaculoviruses (Epap48 and Epap95) are incorporated into OBs. Moreover, quantification based on emPAI values showed that Epap95 is one of the most abundant components of EpapGV OBs.

Long-term preservation of potato leafroll virus, potato virus S, and potato spindle tuber viroid in cryopreserved shoot tips.

Availability of and easy access to diverse plant viruses and viroids is a prerequisite in applied and basic studies related to viruses and viroids. Long-term preservation of viruses and viroids is difficult. A protocol was described for long-term preservation of potato leafroll virus (PLRV), potato virus S (PVS), and potato spindle tuber viroid (PSTVd) in cryopreserved shoot tips of potato cv. Zihuabai. Shoot regrowth levels following cryopreservation were higher in 1.5 mm-shoot tips (58-60%) than in 0.5-mm-ones (30-38%). All shoots recovered from 0.5-mm-shoot tips were PVS- and PSTVd-preserved, but none of them were PLRV-preserved. Cryopreservation of 1.5-mm-shoot tips resulted in 35% and 100% of PLRV- and PVS- and PSTVd-preserved shoots. Studies on cell survival patterns and virus localization provided explanations to the varying PLRV-preservation frequencies produced by cryopreservation of the two sizes of shoot tips. Although micropropagation efficiencies were low after 12 weeks of subculture following cryopreservation, similar efficiencies were obtained after 16 weeks of subculture in pathogen-preserved shoots recovered from cryopreservation, compared with the diseased in vitro stock shoots (the control). Pathogen concentrations in the three pathogens-preserved shoots analyzed by qRT-PCR were similar to those in micropropagated shoots. The three pathogens cryopreserved in shoot tips were readily transmitted by grafting and mechanical inoculation to potato plants. PLRV, PVS, and PSTVd represent a diverse range of plant viruses and viroid in terms of taxonomy and infectious ability. Therefore, shoot tip cryopreservation opens a new avenue for long-term preservation of the virus and viroid.

Virus infection reduces shoot proliferation of in vitro stock cultures and ability of cryopreserved shoot tips to regenerate into normal shoots in 'Gala' apple (Malus × domestica).

Plant cryopreservation has provide secure back-ups of germplasm collections of vegetatively propagated crops. Often, recovery levels vary among laboratories when the same cryogenic procedures are used for the same genotypes. The present study investigated the effects of Apple stem grooving virus (ASGV) on shoot proliferation of in vitro stock cultures and recovery of cryopreserved shoot tips of 'Gala' apple. Results showed that virus infection reduced shoot proliferation of in vitro stock cultures and cell ability to regenerate normal shoots in cryopreserved shoot tips. Virus infection increased total soluble protein, total soluble sugar and free proline levels and altered endogenous levels of indoleacetic acid (IAA) and zeatin riboside (ZR), but induced severe cell membrane damage and caused alternation in mitochondria shape of the in vitro stock shoots. The altered levels of IAA and ZR were most likely to be responsible for the reduced shoot proliferation of in vitro stock culture. Cell damage and alternations in mitochondria shape in ASGV-infected shoot tips were most likely responsible for the reduced cell ability to regenerate normal shoots following cryopreservation. To the best of our knowledge, this is the first study on effects of virus infection on recovery of cryopreserved shoot tips. Results reported here emphasize that healthy in vitro stock cultures should be used for cryopreservation.

Cryotherapy: A Novel Method for Virus Eradication in Economically Important Plant Species.

Virus diseases have been a great threat to production of economically important crops. In practice, the use of virus-free planting material is an effective strategy to control viral diseases. Cryotherapy, developed based on cryopreservation, is a novel plant biotechnology tool for virus eradication. Comparing to the traditional meristem culture for virus elimination, cryotherapy resulted in high efficiency of pathogen eradication. In general, cryotherapy includes seven major steps: (1) introduction of infected plant materials into in vitro cultures, (2) shoot tip excision, (3) tolerance induction of explants to dehydration and subsequent freezing in liquid nitrogen (LN), (4) a short-time treatment of explants in LN, (5) warming and post-culture for regeneration, (6) re-establishment of regenerated plants in greenhouse conditions, and (7) virus indexing.

Cryotherapy by encapsulation-dehydration is effective for in vitro eradication of latent viruses from 'Marubakaido' apple rootstock.

Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) are several major viral pathogens of apple trees, responsible for substantial damage to the world's apple industry. This study aimed to evaluate the effectiveness of the encapsulation-dehydration cryopreservation technique to eradicate these viral pathogens from in vitro shoot tips excised from 'Marubakaido' apple rootstock cultures. Axillary shoot tips were excised from in vitro cultures, encapsulated in alginate beads, precultured in MS salts, dehydrated in a laminar flow hood, immersed in liquid nitrogen, then warmed and recovered on medium. After LN exposure, in vitro rooting and acclimatization, recovered 'Marubakaido' plants exhibited 52% survival and 35% regrowth without callus formation. After 8 months of regrowth, PCR analyses revealed that all the plants were free of ACLSV and ASPV, but 2 out of 20 recovered plants were still infected with ASGV. This is the first report in Brazil of the application of cryotherapy to eradicate viral complexes in Malus. Cryotherapy can facilitate the production of virus-free plants by producing high quality plant material.

Identification of virus-derived siRNAs and their targets in RBSDV-infected rice by deep sequencing.

RNA interference (RNAi) is a conserved mechanism against viruses in plants and animals. It is thought to inactivate the viral genome by producing virus-derived small interfering RNAs (vsiRNAs). Rice black-streaked dwarf virus (RBSDV) is transmitted to plants by the small brown planthopper (Laodelphax striatellus), and seriously threatens production of rice in East Asia, particularly Oryza sativa japonica subspecies. Through deep sequencing, genome-wide comparisons of RBSDV-derived vsiRNAs were made between the japonica variety Nipponbare, and the indica variety 9311. Four small RNA libraries were constructed from the leaves and shoots of each variety. We found 659,756 unique vsiRNAs in the four samples, and only 43,485 reads were commonly shared. The size distributions of vsiRNAs were mostly 21- and 22-nt long, and A/U bias (66-68%) existed at the first nucleotide of vsiRNAs. Additionally, vsiRNAs were continuously but heterogeneously distributed along S1-S10 segments of the RBSDV genome. Distribution profiles of vsiRNA hotspots were similar in different hosts and tissues, and the 5'- and 3'-terminal regions of S4, S5, and S8 had more hotspots. Distribution and abundance of RBSDV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. Degradome analysis found 25 and 11 host genes appeared to be targeted by vsiRNAs in 9311 and Nipponbare. We report for the first time vsiRNAs derived from RBSDV-infected rice.

Combining Thermotherapy with Cryotherapy for Efficient Eradication of Apple stem grooving virus from Infected In-vitro-cultured Apple Shoots.

Apple stem grooving virus (ASGV), a difficult-to-eradicate virus from apple propagative materials, causes serious damage to apple production. The use of virus-free plants has been and is an effective strategy for control of plant viral diseases. This study aimed to eradicate ASGV from virus-infected in-vitro-cultured shoots of four apple cultivars and one rootstock by combining thermotherapy with cryotherapy. In vitro stock shoots infected with ASGV were thermo-treated using an alternating temperature of 36°C (day) and 32°C (night). Shoot tips were excised from the treated stock shoots and subjected to cryotherapy. Results showed that, although thermotherapy did not influence shoot survival rates, it reduced shoot growth and proliferation of in vitro shoots. Shoot regrowth rates decreased while virus eradication frequencies increased in cryo-treated shoot tips as time durations of thermotherapy increased from 0 to 6 weeks. Shoot regrowth and frequency of virus eradication were positively and negatively correlated, respectively, with the size of shoot tips. The protocol established here yielded shoot regrowth rates and virus eradication frequencies of 33 to 76% and 30 to 100%, respectively, in the four apple cultivars and one rootstock. Thermotherapy altered virus distribution patterns, subsequently resulting in production of a larger virus-free area in the thermo-treated shoot tips. Many cells in the top layers of apical dome and some cells in the youngest leaf primordia survived in cryo-treated shoot tips; these cells were most likely free of virus infection. Thus, plants regenerated from the procedure of combining thermotherapy with cryotherapy were free of ASGV, as judged by reverse-transcription polymerase chain reaction. To the best of our knowledge, this is the widest-spectrum technique reported thus far for the production of ASGV-free plants and provides a novel biotechnology for the production of virus-free plants in Malus spp.

Sequence Exchange between Homologous NB-LRR Genes Converts Virus Resistance into Nematode Resistance, and Vice Versa.

Plants have evolved a limited repertoire of NB-LRR disease resistance (R) genes to protect themselves against myriad pathogens. This limitation is thought to be counterbalanced by the rapid evolution of NB-LRR proteins, as only a few sequence changes have been shown to be sufficient to alter resistance specificities toward novel strains of a pathogen. However, little is known about the flexibility of NB-LRR R genes to switch resistance specificities between phylogenetically unrelated pathogens. To investigate this, we created domain swaps between the close homologs Gpa2 and Rx1, which confer resistance in potato (Solanum tuberosum) to the cyst nematode Globodera pallida and Potato virus X, respectively. The genetic fusion of the CC-NB-ARC of Gpa2 with the LRR of Rx1 (Gpa2CN/Rx1L) results in autoactivity, but lowering the protein levels restored its specific activation response, including extreme resistance to Potato virus X in potato shoots. The reciprocal chimera (Rx1CN/Gpa2L) shows a loss-of-function phenotype, but exchange of the first three LRRs of Gpa2 by the corresponding region of Rx1 was sufficient to regain a wild-type resistance response to G. pallida in the roots. These data demonstrate that exchanging the recognition moiety in the LRR is sufficient to convert extreme virus resistance in the leaves into mild nematode resistance in the roots, and vice versa. In addition, we show that the CC-NB-ARC can operate independently of the recognition specificities defined by the LRR domain, either aboveground or belowground. These data show the versatility of NB-LRR genes to generate resistance to unrelated pathogens with completely different lifestyles and routes of invasion.

Cleaning cassava genotypes infected with cassava frogskin disease via in vitro shoot tip culture.

This study aimed to develop a methodology for eliminating cassava frogskin disease (CFSD) from in vitro shoot tip culture by associating thermotherapy and tetracycline. Cuttings from different accessions (BGM0232, BGM0315, BGM0464, BGM584, BGM0841, and BGM1342), infected with CFSD according to visual inspection of the disease symptoms, were used for cleaning. To verify the absence of other diseases, the plants were indexed for Cassava common mosaic virus - CsCMV (by ELISA) and Cassava vein mosaic virus - CsVMV (by polymerase chain reaction, PCR), proving that the accessions were free of these viruses, except for BGM0315 and BGM0464, which were infected with CsVMV. Subsequently, the cuttings were submitted to different tetracycline concentrations for 3 min, and then subjected to thermotherapy under different temperatures (35°, 38°, 40°, 45°, and 55°C). Shoots of 2 cm were harvested, and their surfaces were sterilized in a laminar flow chamber. Subsequently, the shoot tips of different sizes were removed (0.2, 0.4, 0.5, and 1.0 mm) for inoculation in a culture medium with tetracycline at the same concentrations in which the cuttings were dipped. After 60 days of cultivation, the explants were transferred to a multiplication medium without antibiotics. Thirty days after the transfer, the viability of the regenerated plants was evaluated, which were then acclimatized for 70 days in a greenhouse and transferred to the field. After 7 months, a visual analysis of the symptomatic roots and a PCR analysis were held to prove the elimination of CFSD and CsVMV from the accessions infected with these viruses (BGM0315 and BGM0464), respectively. Most of the treatments resulted in 100% cleaning of CFSD-infected plants. From accessions that were also infected with CsVMV, only 2% of the plants remained infected, also demonstrating the cleaning efficiency of this protocol for this disease.

Infection of non-host model plant species with the narrow-host-range Cacao swollen shoot virus.

Cacao swollen shoot virus (CSSV) is a major pathogen of cacao (Theobroma cacao) in Africa, and long-standing efforts to limit its spread by the culling of infected trees have had very limited success. CSSV is a particularly difficult virus to study, as it has a very narrow host range, limited to several tropical tree species. Furthermore, the virus is not mechanically transmissible, and its insect vector can only be used with difficulty. Thus, the only efficient means to infect cacao plants that have been experimentally described so far are by particle bombardment or the agroinoculation of cacao plants with an infectious clone. We have genetically transformed three non-host species with an infectious form of the CSSV genome: two experimental hosts widely used in plant virology (Nicotiana tabacum and N. benthamiana) and the model species Arabidopsis thaliana. In transformed plants of all three species, the CSSV genome was able to replicate, and, in tobacco, CSSV particles could be observed by immunosorbent electron microscopy, demonstrating that the complete virus cycle could be completed in a non-host plant. These results will greatly facilitate the preliminary testing of CSSV control strategies using plants that are easy to raise and to transform genetically.

The Agrobacterium rhizogenes oncogenes rolB and ORF13 increase formation of generative shoots and induce dwarfism in Arabidopsis thaliana (L.) Heynh.

Plant transformation with the wild type Ri plasmid T-DNA of Agrobacterium rhizogenes is a promising method for breeding of compact plants and has been the subject of numerous studies. However, knowledge concerning the isolated functions of single genes and ORFs from the plasmid is limited. The rolB and ORF13 oncogenes of A. rhizogenes show considerable promise in plant breeding, but have not been comprehensively studied. Detailed information regarding the morphological impact of specific genes of the Ri plasmid will allow for optimized targeted breeding of plants transformed with the wild type Ri plasmid T-DNA. rolB and ORF13 were recombined into the genome of Arabidopsis thaliana using Gateway® cloning and the effect on plant growth was assessed biometrically throughout the plants' life cycle. rolB-lines exhibited dwarfing, early necrosis of rosette leaves, altered leaf and flower morphology, and developed an increased number of inflorescences per rosette area compared to the wild type. ORF13-lines were extremely dwarfed, attaining only ca. 1% of the rosette area of the wild type, leaf and flower size was reduced, and the shape modified. The study documents that the traits inferred by the rolB oncogene yield plants with increased formation of generative shoots, but also result in some degree of premature senescence of vegetative organs. The extreme dwarfism seen in ORF13-lines indicate that this oncogene may be more important in the dwarfing response of plants transformed with the wild type Ri plasmid T-DNA than previously assumed and that transformation with this oncogene induces a very compact phenotype.

Characterization of virus-derived small interfering RNAs in Apple stem grooving virus-infected in vitro-cultured Pyrus pyrifolia shoot tips in response to high temperature treatment.

BACKGROUND: Heat treatment (known as thermotherapy) together with in vitro culture of shoot meristem tips is a commonly used technology to obtain virus-free germplasm for the effective control of virus diseases in fruit trees. RNA silencing as an antiviral defense mechanism has been implicated in this process. To understand if high temperature-mediated acceleration of the host antiviral gene silencing system in the meristem tip facilitates virus-derived small interfering RNAs (vsiRNA) accumulation to reduce the viral RNA titer in the fruit tree meristem tip cells, we used the Apple stem grooving virus (ASGV)-Pyrus pyrifolia pathosystem to explore the possible roles of vsiRNA in thermotherapy. RESULTS: At first we determined the full-length genome sequence of the ASGV-Js2 isolate and then profiled vsiRNAs in the meristem tip of in vitro-grown pear (cv. 'Jinshui no. 2') shoots infected by ASGV-Js2 and cultured at 24 and 37 °C. A total of 7,495 and 7,949 small RNA reads were obtained from the tips of pear shoots cultured at 24 and 37 °C, respectively. Mapping of the vsiRNAs to the ASGV-Js2 genome revealed that they were unevenly distributed along the ASGV-Js2 genome, and that 21- and 22-nt vsiRNAs preferentially accumulated at both temperatures. The 5'-terminal nucleotides of ASGV-specific siRNAs in the tips cultured under different temperatures had a similar distribution pattern, and the nucleotide U was the most frequent. RT-qPCR analyses suggested that viral genome accumulation was drastically compromised at 37 °C compared to 24 °C, which was accompanied with the elevated levels of vsiRNAs at 37 °C. As plant Dicer-like proteins (DCLs), Argonaute proteins (AGOs), and RNA-dependent RNA polymerases (RDRs) are implicated in vsiRNA biogenesis, we also cloned the partial sequences of PpDCL2,4, PpAGO1,2,4 and PpRDR1 genes, and found their expression levels were up-regulated in the ASGV-infected pear shoots at 37 °C. CONCLUSIONS: Collectively, these results showed that upon high temperature treatment, the ASGV-infected meristem shoot tips up-regulated the expression of key genes in the RNA silencing pathway, induced the biogenesis of vsiRNAs and inhibited viral RNA accumulation. This study represents the first report on the characterization of the vsiRNA population in pear plants infected by ASGV-Js2, in response to high temperature treatment.