A VIGS screen identifies immunity in the Arabidopsis Pla-1 accession to viruses in two different genera of the Geminiviridae.

Geminiviruses are DNA viruses that cause severe crop losses in different parts of the world, and there is a need for genetic sources of resistance to help combat them. Arabidopsis has been used as a source for virus-resistant genes that derive from alterations in essential host factors. We used a virus-induced gene silencing (VIGS) vector derived from the geminivirus Cabbage leaf curl virus (CaLCuV) to assess natural variation in virus-host interactions in 190 Arabidopsis accessions. Silencing of CH-42, encoding a protein needed to make chlorophyll, was used as a visible marker to discriminate asymptomatic accessions from those showing resistance. There was a wide range in symptom severity and extent of silencing in different accessions, but two correlations could be made. Lines with severe symptoms uniformly lacked extensive VIGS, and lines that showed attenuated symptoms over time (recovery) showed a concomitant increase in the extent of VIGS. One accession, Pla-1, lacked both symptoms and silencing, and was immune to wild-type infectious clones corresponding to CaLCuV or Beet curly top virus (BCTV), which are classified in different genera in the Geminiviridae. It also showed resistance to the agronomically important Tomato yellow leaf curl virus (TYLCV). Quantitative trait locus mapping of a Pla-1 X Col-0 F2 population was used to detect a major peak on chromosome 1, which is designated gip-1 (geminivirus immunity Pla-1-1). The recessive nature of resistance to CaLCuV and the lack of obvious candidate genes near the gip-1 locus suggest that a novel resistance gene(s) confers immunity.

Virus-induced gene silencing of fiber-related genes in cotton.

Virus-Induced Gene Silencing (VIGS) is a useful method for transient downregulation of gene expression in crop plants. The geminivirus Cotton leaf crumple virus (CLCrV) has been modified to serve as a VIGS vector for persistent gene silencing in cotton. Here the use of Green Fluorescent Protein (GFP) is described as a marker for identifying silenced tissues in reproductive tissues, a procedure that requires the use of transgenic plants. Suggestions are given for isolating and cloning combinations of target and marker sequences so that the total length of inserted foreign DNA is between 500 and 750 bp. Using this strategy, extensive silencing is achieved with only 200-400 bp of sequence homologous to an endogenous gene, reducing the possibility of off-target silencing. Cotyledons can be inoculated using either the gene gun or Agrobacterium and will continue to show silencing throughout fruit and fiber development. CLCrV is not transmitted through seed, and VIGS is limited to genes expressed in the maternally derived seed coat and fiber in the developing seed. This complicates the use of GFP as a marker for VIGS because cotton fibers must be separated from unsilenced tissue in the seed to determine if they are silenced. Nevertheless, fibers from a large number of seeds can be rapidly screened following placement into 96-well plates. Methods for quantifying the extent of silencing using semiquantitative RT-PCR are given.

Persistent virus-induced gene silencing in asymptomatic accessions of Arabidopsis.

Coupled with the advantages afforded by the model plant Arabidopsis, virus-induced gene silencing (VIGS) offers a rapid means to assess gene function. The geminivirus vector based on Cabbage leaf curl virus described here has the benefits of small insert size and persistent silencing of the target gene through the life cycle of the plant. Here, we show that genetic variation in the vast collection of Arabidopsis accessions can be leveraged to ameliorate viral symptomology that accompanies the VIGS procedure. The plasticity of phenotypes under different day lengths or temperature conditions can be exploited to achieve maximum silencing efficacy in either vegetative or inflorescence tissue, according to the question being asked. Protocols and vectors for Agro-infiltration of primary leaves, subapical pricking in older plants, and microprojectile bombardment are described.

Geminiviruses: masters at redirecting and reprogramming plant processes.

The family Geminiviridae is one of the largest and most important families of plant viruses. The small, single-stranded DNA genomes of geminiviruses encode 5-7 proteins that redirect host machineries and processes to establish a productive infection. These interactions reprogramme plant cell cycle and transcriptional controls, inhibit cell death pathways, interfere with cell signalling and protein turnover, and suppress defence pathways. This Review describes our current knowledge of how geminiviruses interact with their plant hosts and the functional consequences of these interactions.

A new virus-induced gene silencing vector based on Euphorbia mosaic virus-Yucatan peninsula for NPR1 silencing in Nicotiana benthamiana and Capsicum annuum var. Anaheim.

Virus-induced gene silencing is based on the sequence-specific degradation of RNA. Here, a gene silencing vector derived from EuMV-YP, named pEuMV-YP:ΔAV1, was used to silence ChlI and NPR1 genes in Nicotiana benthamiana. The silencing of the ChlI transcripts was efficient in the stems, petioles and leaves as reflected in tissue bleaching and reduced transcript levels. The silencing was stable, reaching the flowers and fruits, and was observed throughout the life cycle of the plants. Additionally, the silencing of the NPR1 gene was efficient in both N. benthamiana and Capsicum annuum. After silencing, the plants' viral symptoms increased to levels similar to those seen in wild-type plants. These results suggest that NPR1 plays a role in the compatible interactions of EuMV-YP N. benthamiana and EuMV-C. annum var. anaheim.

Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system.

BACKGROUND: We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns. RESULTS: The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively. CONCLUSIONS: These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.

An ER-targeted calcium-binding peptide confers salt and drought tolerance mediated by CIPK6 in Arabidopsis.

Different plant organelles have high internal stores of Ca(2+) compared to the cytoplasm and could play independent roles in stress responses or signal transduction. We used a GFP fusion with the C-domain of calreticulin, which shows low-affinity, high capacity Ca(2+) binding in the ER, as a calcium-binding peptide (CBP) to specifically increase stores in the ER and nucleus. Despite the presence of a signal sequence and KDEL retention sequence, our work and previous studies (Brandizzi et al. Plant Journal 34:269-281, 2003) demonstrated both ER and nuclear localization of GFP-CBP. Under normal conditions, GFP-CBP-expressing lines had ~25% more total Ca(2+) and higher levels of chlorophyll and seed yield than wild type and GFP controls. CBP-expressing plants also had better survival under intermittent drought or high salt treatments and increased root growth. One member of the CIPK (calcineurin B-like interacting protein kinase) gene family, CIPK6, was up-regulated in CBP-expressing plants, even under non-stress conditions. A null mutation in cipk6 abolished the increased stress tolerance of CBP-transgenic plants, as well as the CBP-mediated induction of two stress-associated genes, DREB1A and RD29A, under non-stress conditions. Although this suggested that it was the induction of CIPK6, rather than localized changes in Ca(2+), that resulted in increased survival under adverse conditions, CIPK6 induction still required Ca(2+). This work demonstrates that ER (or nuclear) Ca(2+) can directly participate in signal transduction to alter gene expression. The discovery of a method for increasing Ca(2+) levels without deleterious effects on plant growth may have practical applications.

The effect of temperature on Natural Antisense Transcript (NAT) expression in Aspergillus flavus.

Naturally occurring Antisense Transcripts (NATs) compose an emerging group of regulatory RNAs. These regulatory elements appear in all organisms examined, but little is known about global expression of NATs in fungi. Analysis of currently available EST sequences suggests that 352 cis NATs are present in Aspergillus flavus. An Affymetrix GeneChip microarray containing probes for these cis NATs, as well as all predicted genes in A. flavus, allowed a whole genome expression analysis of these elements in response to two ecologically important temperatures for the fungus. RNA expression analysis showed that 32 NATs and 2,709 genes were differentially expressed between 37 degrees C, the optimum temperature for growth, and 28 degrees C, the conducive temperature for the biosynthesis of aflatoxin (AF) and many other secondary metabolites. These NATs correspond to sense genes with diverse functions including transcription initiation, carbohydrate processing and binding, temperature sensitive morphogenesis, and secondary metabolism. This is the first report of a whole genome transcriptional analysis of NAT expression in a fungus.

Geminivirus-mediated gene silencing from Cotton leaf crumple virus is enhanced by low temperature in cotton.

A silencing vector for cotton (Gossypium hirsutum) was developed from the geminivirus Cotton leaf crumple virus (CLCrV). The CLCrV coat protein gene was replaced by up to 500 bp of DNA homologous to one of two endogenous genes, the magnesium chelatase subunit I gene (ChlI) or the phytoene desaturase gene (PDS). Cotyledons of cotton cultivar 'Deltapine 5415' bombarded with the modified viral vectors manifested chlorosis due to silencing of either ChlI or PDS in approximately 70% of inoculated plants after 2 to 3 weeks. Use of the green fluorescence protein gene showed that replication of viral DNA was restricted to vascular tissue and that the viral vector could transmit to leaves, roots, and the ovule integument from which fibers originate. Temperature had profound effects on vector DNA accumulation and the spread of endogenous gene silencing. Consistent with reports that silencing against viruses increases at higher temperatures, plants grown at a 30 degrees C/26 degrees C day/night cycle had a greater than 10-fold reduction in viral DNA accumulation compared to plants grown at 22 degrees C/18 degrees C. However, endogenous gene silencing decreased at 30 degrees C/26 degrees C. There was an approximately 7 d delay in the onset of gene silencing at 22 degrees C/18 degrees C, but silencing was extensive and persisted throughout the life of the plant. The extent of silencing in new growth could be increased or decreased by changing temperature regimes at various times following the onset of silencing. Our experiments establish the use of the CLCrV silencing vector to study gene function in cotton and show that temperature can have a major impact on the extent of geminivirus-induced gene silencing.

Geminivirus-induced gene silencing of the tobacco retinoblastoma-related gene results in cell death and altered development.

The retinoblastoma-related protein (RBR) is required for cell cycle control and differentiation and is expressed throughout the life of plants and animals. In this study, the tomato golden mosaic virus (TGMV) geminivirus vector was used to silence NbRBR1 in Nicotiana benthamiana by microprojectile bombardment into fully developed leaves. Similar to previous results using agroinoculation of a tobacco rattle virus silencing vector [Park et al. (Plant J 42:153, 2005)], developmental defects caused by disruptions in cell size and number were seen in new growth. Leaf midvein cross-sections showed tissue-specific differences in size, cell number, and cell morphology. While cortical cell numbers decreased, size increased to maintain overall shape. In contrast, xylem parenchyma cells increased approximately three fold but remained small. Normally straight flowers often curved up to 360 degrees without a significant change in size. However, the most striking phenotype was cell death in mature cells after a delay of 3-4 weeks. Trypan blue staining confirmed cell death and demonstrated that cell death was absent in similarly treated leaves of wild type TGMV-inoculated plants. Quantitative RT-PCR confirmed that the mature TGMV:RBR-inoculated leaves still maintained reduced accumulation of RBR transcript at 4 weeks compared to controls. The results suggest that either inappropriate activation of the cell cycle is lethal in plants or that RBR has other functions, unrelated to the cell cycle. The results also demonstrate that continual transcription of RBR is necessary for cell survival.

Silencing of the aflatoxin gene cluster in a diploid strain of Aspergillus flavus is suppressed by ectopic aflR expression.

Aflatoxins are toxic secondary metabolites produced by a 70-kb cluster of genes in Aspergillus flavus. The cluster genes are coordinately regulated and reside as a single copy within the genome. Diploids between a wild-type strain and a mutant (649) lacking the aflatoxin gene cluster fail to produce aflatoxin or transcripts of the aflatoxin pathway genes. This dominant phenotype is rescued in diploids between a wild-type strain and a transformant of the mutant containing an ectopic copy of aflR, the transcriptional regulator of the aflatoxin biosynthetic gene cluster. Further characterization of the mutant showed that it is missing 317 kb of chromosome III, including the known genes for aflatoxin biosynthesis. In addition, 939 kb of chromosome II is present as a duplication on chromosome III in the region previously containing the aflatoxin gene cluster. The lack of aflatoxin production in the diploid was not due to a unique or a mis-expressed repressor of aflR. Instead a form of reversible silencing based on the position of aflR is likely preventing the aflatoxin genes from being expressed in 649 x wild-type diploids. Gene expression analysis revealed the silencing effect is specific to the aflatoxin gene cluster.

Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing.

Like other eukaryotes, plants use DICER-LIKE (DCL) proteins as the central enzymes of RNA silencing, which regulates gene expression and mediates defense against viruses. But why do plants like Arabidopsis express four DCLs, a diversity unmatched by other kingdoms? Here we show that two nuclear DNA viruses (geminivirus CaLCuV and pararetrovirus CaMV) and a cytoplasmic RNA tobamovirus ORMV are differentially targeted by subsets of DCLs. DNA virus-derived small interfering RNAs (siRNAs) of specific size classes (21, 22 and 24 nt) are produced by all four DCLs, including DCL1, known to process microRNA precursors. Specifically, DCL1 generates 21 nt siRNAs from the CaMV leader region. In contrast, RNA virus infection is mainly affected by DCL4. While the four DCLs are partially redundant for CaLCuV-induced mRNA degradation, DCL4 in conjunction with RDR6 and HEN1 specifically facilitates extensive virus-induced silencing in new growth. Additionally, we show that CaMV infection impairs processing of endogenous RDR6-derived double-stranded RNA, while ORMV prevents HEN1-mediated methylation of small RNA duplexes, suggesting two novel viral strategies of silencing suppression. Our work highlights the complexity of virus interaction with host silencing pathways and suggests that DCL multiplicity helps mediate plant responses to diverse viral infections.

VIGS vectors for gene silencing: many targets, many tools.

The discovery that plants recognize and degrade invading viral RNA caused a paradigm shift in our understanding of viral/host interactions. Combined with the discovery that plants cosuppress their own genes if they are transformed with homologous transgenes, new models for both plant intercellular communication and viral defense have emerged. Plant biologists adapted homology-based defense mechanisms triggered by incoming viruses to target individual genes for silencing in a process called virus-induced gene silencing (VIGS). Both VIGS- and dsRNA-containing transformation cassettes are increasingly being used for reverse genetics as part of an integrated approach to determining gene function. Virus-derived vectors silence gene expression without transformation and selection. However, because viruses also alter gene expression in their host, the process of VIGS must be understood. This review examines how DNA and RNA viruses have been modified to silence plant gene expression. I discuss advantages and disadvantages of VIGS in determining gene function and guidelines for the safe use of viral vectors.

Geminivirus VIGS of endogenous genes requires SGS2/SDE1 and SGS3 and defines a new branch in the genetic pathway for silencing in plants.

Virus-induced gene silencing (VIGS) is a sequence-specific RNA degradation process that can be used to downregulate plant gene expression. Both RNA and DNA viruses have been used for VIGS, but they differ in their mode of replication, gene expression, and cellular location. This study examined silencing mediated by a DNA virus, cabbage leaf curl virus (CaLCuV), in several silencing-deficient Arabidopsis mutants. A DNA VIGS vector derived from CaLCuV, which silenced chlorata42 (ChlI) needed for chlorophyll formation, was used to test endogenous gene silencing responses in suppressor of gene silencing (sgs)1, sgs2, sgs3, and Argonaute (ago)1 mutants defective in sense transgene-mediated post-transcriptional silencing (S-PTGS). SGS2/silencing defective (SDE)1, SGS3, and AGO1 are each dispensable for silencing mediated by transgenes containing inverted repeats (IR-PTGS), and SGS2/SDE1 is dispensable for RNA VIGS. We show that DNA VIGS requires both SGS2/SDE1 and SGS3, regardless of the orientation of 362 nt ChlI transcripts produced from the viral DNA promoter. Viral DNA accumulation is slightly higher, and viral symptoms increase in sgs2 and sgs3, whereas overexpression of SGS2/SDE1 mRNA results in decreased viral symptoms. Mutants affected in SGS1 and AGO1 function are only delayed in the onset of silencing, and have a small effect on chlorophyll accumulation. DNA VIGS is unaffected in defective DNA methylation (ddm)1/somniferous (som)8 and maintenance of methylation (mom)1 mutants, impaired for TGS. These results demonstrate that SGS2/SDE1 and SGS3 are needed for endogenous gene silencing from DNA viruses, and suggest that SGS2/SDE1 may reduce geminivirus symptoms by targeting viral mRNAs.

Geminivirus vectors for transient gene silencing in plants.

Both RNA and DNA viruses have been engineered to serve as vectors for transient silencing in intact plants. Host gene sequences carried by the virus are seen by the plant as "foreign," and homologous gene-silencing machinery acts on both the viral vector RNA and the endogenous host gene mRNA. DNA viruses, such as geminiviruses, are advantageous for silencing because only their mRNAs are silenced and their DNA genomes continue to replicate and move. The conserved genome organization of geminiviruses and the fact that they can be cloned into Escherichia coli plasmids, propagated, and then inoculated into plants for infection simplifies the procedure for silencing specific chromosomal genes in intact plants. This chapter describes the development of a silencing vector from cabbage leaf curl virus for use in Arabidopsis and procedures for silencing two genes simultaneously.

Reprogramming plant gene expression: a prerequisite to geminivirus DNA replication.

SUMMARY Geminiviruses constitute a large family of plant-infecting viruses with small, single-stranded DNA genomes that replicate through double-stranded intermediates. Because of their limited coding capacity, geminiviruses supply only the factors required to initiate their replication and use plant nuclear DNA polymerases to amplify their genomes. Many geminiviruses replicate in differentiated cells that no longer contain detectable levels of host DNA polymerases and associated factors. To overcome this barrier, geminiviruses induce the accumulation of DNA replication machinery in mature plant cells by reprogramming host gene expression. The mammalian DNA tumour viruses activate host genes required for DNA replication by binding to the retinoblastoma protein, a negative regulator of cell cycle progression, and relieving repression through the E2F family of transcription factors. In this review, we discuss recent experiments showing that geminiviruses also modulate components of the retinoblastoma/E2F transcription regulatory network to induce quiescent plant cells to re-enter the cell cycle and regain the capacity to support high levels of DNA replication. Regulation of the cell division cycle and its integration with developmental pathways is complex, with many factors, including hormones, sucrose and environmental signals, controlling re-entry into the plant cell cycle. Geminivirus interactions with these regulatory networks are likely to determine if and where they can replicate their genomes in different plant tissues and hosts.

Host DNA replication is induced by geminivirus infection of differentiated plant cells.

The geminivirus Tomato golden mosaic virus (TGMV) replicates in differentiated plant cells using host DNA synthesis machinery. We used 5-bromo-2-deoxyuridine (BrdU) incorporation to examine DNA synthesis directly in infected Nicotiana benthamiana plants to determine if viral reprogramming of host replication controls had an impact on host DNA replication. Immunoblot analysis revealed that up to 17-fold more BrdU was incorporated into chromosomal DNA of TGMV-infected versus mock-infected, similarly treated healthy leaves. Colocalization studies of viral DNA and BrdU demonstrated that BrdU incorporation was specific to infected cells and was associated with both host and viral DNA. TGMV and host DNA synthesis were inhibited differentially by aphidicolin but were equally sensitive to hydroxyurea. Short BrdU labeling times resulted in some infected cells showing punctate foci associated with host DNA. Longer periods showed BrdU label uniformly throughout host DNA, some of which showed condensed chromatin, only in infected nuclei. By contrast, BrdU associated with viral DNA was centralized and showed uniform, compartmentalized labeling. Our results demonstrate that chromosomal DNA is replicated in TGMV-infected cells.

Two E2F elements regulate the proliferating cell nuclear antigen promoter differently during leaf development.

E2F transcription factors regulate genes expressed at the G1/S boundary of the cell division cycle in higher eukaryotes. Although animal E2F proteins and their target promoters have been studied extensively, little is known about how these factors regulate plant promoters. An earlier study identified two E2F consensus binding sites in the promoter of a Nicotiana benthamiana gene encoding proliferating cell nuclear antigen (PCNA) and showed that the proximal element (E2F2) is required for the full repression of PCNA expression in mature leaves. In this study, we examined the distal element (E2F1) and how it interacts with the E2F2 site to regulate the PCNA promoter. Gel shift assays using plant nuclear extracts or purified Arabidopsis E2F and DP proteins showed that different complexes bind to the two E2F sites. Mutation of the E2F1 site or both sites differentially altered PCNA promoter function in transgenic plants. As reported previously for the E2F2 mutation, the E2F1 and E2F1+2 mutations partially relieved the repression of the PCNA promoter in mature leaves. In young tissues, the E2F1 mutation resulted in a threefold reduction in PCNA promoter activity, whereas the E2F1+2 mutation had no detectable effect. The activity of E2F1+2 mutants was indistinguishable from that of E2F2 mutants. These results demonstrate that both E2F elements contribute to the repression of the PCNA promoter in mature leaves, whereas the E2F1 site counters the repression activity of the E2F2 element in young leaves.

Mutations in the gravity persistence signal loci in Arabidopsis disrupt the perception and/or signal transduction of gravitropic stimuli.

Gravity plays a fundamental role in plant growth and development, yet little is understood about the early events of gravitropism. To identify genes affected in the signal perception and/or transduction phase of the gravity response, a mutant screen was devised using cold treatment to delay the gravity response of inflorescence stems of Arabidopsis. Inflorescence stems of Arabidopsis show no response to gravistimulation at 4 degrees C for up to 3 h. However, when gravistimulated at 4 degrees C and then returned to vertical at room temperature (RT), stems bend in response to the previous, horizontal gravistimulation (H. Fukaki, H. Fujisawa, M. Tasaka [1996] Plant Physiology 110: 933-943). This indicates that gravity perception, but not the gravitropic response, occurs at 4 degrees C. Recessive mutations were identified at three loci using this cold effect on gravitropism to screen for gravity persistence signal (gps) mutants. All three mutants had an altered response after gravistimulation at 4 degrees C, yet had phenotypically normal responses to stimulations at RT. gps1-1 did not bend in response to the 4 degrees C gravity stimulus upon return to RT. gps2-1 responded to the 4 degrees C stimulus but bent in the opposite direction. gps3-1 over-responded after return to RT, continuing to bend to an angle greater than wild-type plants. At 4 degrees C, starch-containing statoliths sedimented normally in both wild-type and the gps mutants, but auxin transport was abolished at 4 degrees C. These results are consistent with GPS loci affecting an aspect of the gravity signal perception/transduction pathway that occurs after statolith sedimentation, but before auxin transport.

Geminivirus-based vectors for gene silencing in Arabidopsis.

Gene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable gene-silencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene-replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.