Cauliflower Mosaic Virus Gene VI Research Articles

Mutations within A 35 amino acid region of P6 influence self-association, inclusion body formation, and Caulimovirus infectivity

Cauliflower mosaic virus gene VI product (P6) is an essential protein that forms cytoplasmic, inclusion bodies (IBs). P6 contains four regions involved in self-association, termed D1–D4. D3 binds to D1, along with D4 and contains a spacer region (termed D3b) between two RNA-binding domains. Here we show D3b binds full-length P6 along with D1 and D4. Full-length P6s harboring single amino acid substitutions within D3b showed reduced binding to both D1 and D4. Full-length P6s containing D3b mutations and fused with green fluorescent protein formed inclusion-like bodies (IL-Bs) when expressed in Nicotiana benthamiana leaves. However, mutant P6s with reduced binding to D1 and D4, showed smaller IL-Bs, than wild type. Likewise, viruses containing these mutations showed a decrease in inoculated leaf viral DNA levels and reduced efficiency of systemic infection. These data suggest that mutations influencing P6 self-association alter IB formation and reduce virus infection.

Cauliflower mosaic virus gene VI product N-terminus contains regions involved in resistance-breakage, self-association and interactions with movement protein

Cauliflower mosaic virus (CaMV) gene VI encodes a multifunctional protein (P6) involved in the translation of viral RNA, the formation of inclusion bodies, and the determination of host range. Arabidopsis thaliana ecotype Tsu-0 prevents the systemic spread of most CaMV isolates, including CM1841. However, CaMV isolate W260 overcomes this resistance. In this paper, the N-terminal 110 amino acids of P6 (termed D1) were identified as the resistance-breaking region. D1 also bound full-length P6. Furthermore, binding of W260 D1 to P6 induced higher β-galactosidase activity and better leucine-independent growth in the yeast two-hybrid system than its CM1841 counterpart. Thus, W260 may evade Tsu-0 resistance by mediating P6 self-association in a manner different from that of CM1841. Because Tsu-0 resistance prevents virus movement, interaction of P6 with P1 (CaMV movement protein) was investigated. Both yeast two-hybrid analyses and maltose-binding protein pull-down experiments show that P6 interacts with P1. Although neither half of P1 interacts with P6, the N-terminus of P6 binds P1. Interestingly, D1 by itself does not interact with P1, indicating that different portions of the P6 N-terminus are involved in different activities. The P1–P6 interactions suggest a role for P6 in virus transport, possibly by regulating P1 tubule formation or the assembly of movement complexes.

A Comparative Analysis of the Avirulence and Translational Transactivator Functions of Gene VI of Cauliflower mosaic virus

The primary function associated at present with the gene VI product of Cauliflower mosaic virus (CaMV) is that of a translational transactivator (TAV). In this capacity, it alters the host translational machinery to allow reinitiation of translation of other CaMV genes on the polycistronic 35S RNA of CaMV. In addition, the gene VI protein can elicit a specific type of plant defense response called the hypersensitive response (HR) in Nicotiana edwardsonii. In this study, we have adapted the agroinfiltration technique to compare the sequences of CaMV gene VI required for TAV function and elicitation of HR. To measure the activity of the TAV, we coagroinfiltrated gene VI of CaMV strain W260 with a bicistronic GUS reporter plasmid. TAV function could be assayed 4 days postinfiltration, before the onset of HR in N. edwardsonii. Through the use of the TAV and HR assays, we could show that the TAV functions of gene VI of CaMV strains W260 and D4 were equivalent, but only W260 gene VI elicited HR. A mutational analysis of W260 gene VI showed that the structural requirements for elicitation of HR were much more stringent than those for TAV function. Small deletions from either the 5′ or 3′ end of W260 gene VI abolished its ability to elicit HR, although the TAV function was retained in the mutant. The TAV function could also tolerate a small insertion within gene VI; this insertion abolished the elicitor function. This study provides direct evidence that the TAV function of gene VI is separate from its role as an elicitor of HR.

Systemic Cell Death Is Elicited by the Interaction of a Single Gene in Nicotiana clevelandii and Gene VI of Cauliflower Mosaic Virus

Cauliflower mosaic virus (CaMV) strains D4 and W260 can be distinguished by the type of symptoms they induce in Nicotiana clevelandii and N. edwardsonii. W260 induces systemic cell death in addition to a mosaic symptom in N. clevelandii and a hypersensitive response (HR) in N. edwardsonii, whereas D4 induces a systemic mosaic in both hosts. To determine which W260 genes are responsible for systemic cell death, chimeric viruses were constructed between the D4 and W260 strains. It was found that W260 gene VI was responsible for the elicitation of systemic cell death; previous studies had shown that this same gene elicited HR in N. edwardsonii. An immunological analysis of plants infected with W260 or D4 indicated that the systemic cell death symptom was not associated with enhanced levels of either W260 virions or the W260 gene VI product. To investigate the inheritance of systemic cell death, crosses were made between N. clevelandii and N. bigelovii, a host that reacts with a systemic mosaic symptom upon infection with W260. All F1 plants developed a systemic mosaic after inoculation with W260, whereas the F2 generation segregated 3:1 for systemic mosaic versus cell death. The plant gene responsible for cell death was designated ccd1, for CaMV cell death gene. These results demonstrate that the systemic cell death symptom in N. clevelandii is induced by the interaction between a single host gene and gene VI of CaMV.

Altered patterns of gene expression in Arabidopsis elicited by cauliflower mosaic virus (CaMV) infection and by a CaMV gene VI transgene.

Cauliflower mosaic virus (CaMV) gene VI protein (P6) is an important determinant of symptom expression. Differential display polymerase chain reaction (PCR) was used to identify changes in gene expression in Arabidopsis elicited by a P6 transgene that causes a symptomatic phenotype. We used slot blot hybridization to measure the abundance of mRNAs complementary to 66 candidate PCR products in transgenic, CaMV-infected, and uninfected Arabidopsis plants. CaMV-infected and P6 transgenic plants showed broadly similar changes in abundance of mRNA species. In P6 transgenic plants we detected 18 PCR products that showed unambiguous changes in abundance plus another 15 that showed more limited changes (approximately twofold). CaMV-infected plants showed 17 unambiguous and 13 limited changes. Down-regulated species include those encoding a novel, phenol-like sulfotransferase, and a glycine-rich, RNA-binding protein. Up-regulated species included ones encoding an myb protein, glycine-rich and stress-inducible proteins, and a member of a previously unreported gene family. CaMV infection causes alterations in expression of many Arabidopsis genes. Transgene-mediated expression of P6 mimics virus infection in its effect on host gene expression, providing a potential mechanism for this process.

Light-Dependent Systemic Infection of Solanaceous Species by Cauliflower Mosaic Virus Can Be Conditioned by a Viral Gene Encoding an Aphid Transmission Factor

Gene II of cauliflower mosaic virus (CaMV), which encodes an 18-kDa protein originally identified as an aphid transmission factor (ATF), influences host specificity in a light-dependent manner. A point mutation within the ATF gene that occurs in several CaMV strains was responsible for conditioning light-dependent systemic infections. A point mutant of CaMV strain W260 that carried the single mutation within the ATF gene was able to systemically infectNicotiana bigeloviiat low light intensity (100–180 μmol m−2sec−1), but not at a higher light intensity level (350–450 μmol m−2sec−1), while the wild-type W260 virus could systemically infectN. bigeloviiunder both light conditions. The same point mutation also affected the stability of the amorphous CaMV inclusions and previous studies have shown that it abolishes transmission of CaMV by aphids. The point mutation within the ATF gene that mediated light-dependent infections was complemented by transgenicN. bigeloviiplants that express the CaMV gene VI product, a viral protein that has been identified as a translational transactivator. The complementation studies indicated that the ATF gene may influence systemic infections through an interaction with the CaMV gene VI product. The ATF gene of CaMV may contribute to viral infections by regulating expression of downstream genes or by influencing cell-to-cell or long distance movement within the plant.

Isolation of Recombinant Viruses between Cauliflower Mosaic Virus and a Viral Gene in Transgenic Plants under Conditions of Moderate Selection Pressure

We demonstrate that recombinant viruses formed between a wild-type virus and a viral transgene can be isolated from transgenic plants under conditions of moderate to weak selection pressure. We inoculated cauliflower mosaic virus (CaMV) strain W260 to transgenicNicotiana bigeloviiplants that expressed a copy of CaMV gene VI derived from CaMV strain D4, a gene that determines systemic infection of solanaceous species, includingN. bigelovii.Because W260 infects nontransformedN. bigeloviisystemically, a recombinant virus formed between W260 and the D4 transgene would be expected to have little selective advantage over the wild-type W260 virus. W260 was inoculated to approximately 100 plants each of nontransformed and transgenicN. bigeloviiand it systemically infected nearly all of the plants. An analysis of viral DNA recovered from 23 transgenic plants infected with W260 revealed that 20 infections resulted from the systemic movement of the wild-type W260 virus, while a recombinant between W260 and the D4 transgene was detected in three of the infections. To determine the percentage of recovery of recombinant viruses under strong selection pressure, we inoculated approximately 100 nontransformed and 100 D4 gene VI transgenic plants with CaMV strain CM1841, a virus that is unable to infect nontransformedN. bigelovii.CM1841 infected 36% of the transgenic plants systemically, but none of the nontransformed controls. An analysis of 24 infected plants showed that a recombination event occurred in every plant, demonstrating that under strong selection conditions, the recovery of CaMV recombinants from transgenic plants can be very high.

Expression of a plant viral polycistronic mRNA in yeast, Saccharomyces cerevisiae, mediated by a plant virus translational transactivator.

We demonstrate that the cauliflower mosaic virus (CaMV) gene VI product can transactivate the expression of a reporter gene in bakers' yeast, Saccharomyces cerevisiae. The gene VI coding sequence was placed under the control of the galactose-inducible promoter GAL1, which is presented in the yeast shuttle vector pYES2, to create plasmid JS169. We also created a chloramphenicol acetyltransferase (CAT) reporter plasmid, JS161, by inserting the CAT reporter gene in-frame into CaMV gene II and subsequently cloning the entire CaMV genome into the yeast vector pRS314. When JS161 was transformed into yeast and subsequently assayed for CAT activity, only a very low level of CAT activity was detected in cellular extracts. To investigate whether the CaMV gene VI product would mediate an increase in CAT activity, we cotransformed yeast with JS169 and JS161. Upon induction with galactose, we found that CAT activity in yeast transformed with JS161 and JS169 was about 19 times higher than the level in the transformants that contained only JS161. CAT activity was dependent on the presence of the gene VI protein, because essentially no CAT activity was detected in yeast cells grown in the presence of glucose, which represses expression from the GAL1 promoter. RNase protection assays showed that the gene VI product had no effect on transcription from the 35S RNA promoter, demonstrating that regulation was occurring at the translation level. This yeast system will prove useful for understanding how the gene VI product of CaMV mediates the translation of genes present on a eukaryotic polycistronic mRNA.

Mutational analysis of cauliflower mosaic virus gene VI: changes in host range, symptoms, and discovery of transactivation-positive, noninfectious mutants.

Gene VI of cauliflower mosaic virus (CaMV) has been shown to be a determinant of host specificity of the virus as well as a factor influencing symptom development in infected plants. In addition, it plays a crucial role in viral gene expression through a process of posttranscriptional transactivation. In the present study, linker-insertion mutations within gene VI of a cloned, recombinant cauliflower mosaic virus genome were constructed and tested for infectivity, symptom development on solanaceous plants, and the ability of transactivate viral gene expression. Certain mutations in the first third of the gene resulted in changes in symptoms shown by test plants. Another mutation, also in the first third of the gene, blocked infectivity in the Nicotiana species tested and systemic movement in Datura stramonium. The mutants were also tested in protoplasts for the ability to transactivate virus gene expression. Infectious mutants were invariably positive for transactivation and mutants negative for transactivation were noninfectious. Interestingly, two mutants positive for transactivation were noninfectious, suggesting a second function for gene VI in the infection process. These results further suggest a role for gene VI, as yet not fully understood, in systemic movement of the virus in infected plants.

Point Mutations in Cauliflower Mosaic Virus Gene VI Confer Host-Specific Symptom Changes

Mutants of cauliflower mosaic virus (CaMV) strain D4 have been characterized with regard to host-specific phenotypes that resulted from specific changes in the viral DNA sequence. Both the mutant and the wild-type viruses infect a brassicaceous host, Brassica campestris, systemically, giving indistinguishable symptoms. However, in the solanaceous host Datura stramonium, which was systemically infectible by the wild-type virus, mutants induced necrotic local lesions at 21 degrees C and above, and a veinal necrosis at lower temperatures. The mutants differed from the parental D4 strain by having single base changes in gene VI. The necrotic phenotype could be selected during serial passage of D4 in B. campestris or created by site-directed mutagenesis within the gene VI coding region. Full-length 62-kDa gene VI gene products were detected in extracts of plants infected with the mutant strains, as were two smaller proteins derived from the same coding region. The relationship of the host-specific phenotypes of the mutants to the detected gene VI-encoded proteins is discussed in the context of the natural variation found in this gene.

Cauliflower mosaic virus gene VI causes growth suppression, development of necrotic spots and expression of defence-related genes in transgenic tobacco plants

In order to study possible functions of the inclusion body matrix protein (IBMP) encoded by gene VI of cauliflower mosaic virus (CaMV), the XbaI fragment containing the gene VI of a Japanese strain of CaMV (CaMV S-Japan) was transferred to tobacco plants by Ti mediated transformation. Eight out of 18 kanamycin resistant plants (40%) expressed detectable levels of IBMP. Those transgenic plants expressing IBMP produced leaves with light green color, and their growth was suppressed as compared with control plants. Symptom-like necrotic spots also appeared on the leaves and stems of the mature transgenic plants. Furthermore, in these transgenic plants, pathogenesis-related proteins 1a, 1b and 1c were highly expressed and the activity of 1,3-β-glucanase was increased up to eightfold. From these results, we concluded that expression of the IBMP is associated with symptom development.

Cauliflower mosaic virus gene VI produces a symptomatic phenotype in transgenic tobacco plants

Gene VI of the cauliflower mosaic virus (CaMV) genome encodes a protein (P(66)) in virus-infected plants that accumulates in cytoplasmic inclusion bodies. When a segment of the CaMV genome bearing gene VI is transferred to tobacco plants by the Agrobacterium tumefaciens Ti plasmid, the resulting transgenic plants display viral-like symptoms. Symptoms produced by the DNA from two different viral isolates (CaMV Cabb B-JI and CM1841) were distinct-symptoms from the first were mosaic-like, whereas the other caused uniform bleaching of leaves. That gene VI was responsible for the symptomatic phenotype was demonstrated by showing that symptom production was blocked by deletions and by a frame-shifting linker mutation in gene VI. Furthermore, in primary transformants, there was a strict correlation between the appearance of symptoms and the presence of gene VI product, P(66), detected by immunoblots. Hence, a protein encoded by the CaMV genome produces viral-like symptoms in transgenic tobacco plants.

Co-expression and inheritance of foreign genes in transformants obtained by direct DNA transformation of tobacco protoplasts

A plasmid containing two marker genes for expression in plants was constructed. This 16 kb vector, pCT1T3, contains an intact nopaline synthase gene and a chimaeric gene consisting of the promoter and terminator regions from cauliflower mosaic virus gene VI and a structural gene, aminoglycoside phosphotransferase (APH(3′)II), from the bacterial transposon Tn5. After transformation of tobacco mesophyll protoplasts with this plasmid, several kanamycin-resistant transformants were obtained. Intensive studies on the drug tolerance of growth and differentiation of the transformants showed that the chimaeric gene was stably expressed. Of 17 independent transformants, 3 (about 18%) expressed the two marker genes, regardless of the state of differentiation, as did individual plants regenerated from the same callus. Multiple copies of the inserted DNA were found in some transformants.

Molecular and general genetics of a hybrid foreign gene introduced into tobacco by direct gene transfer.

Two clones of N. tabacum, transformed to kanamycin resistance by direct transfer to protoplasts of a hybrid gene, consisting of the protein coding region from the bacterial gene for aminoglycoside phosphotransferase under the control of 5'/3' expression signals from cauliflower mosaic virus gene VI, in the bacterial plasmid pUC8, have been subjected to a detailed genetic crossing analysis accompanied by Southern blot analysis and enzyme activity assays of representative offspring. The genetic data obtained from large populations of R1/F1 and R2/F2 offspring as well as from more than 20 subclones of each of the original of the hybrid gene was stably integrated into chromosomal DNA of the original transformants, (b) that the gene normally was stably maintained during clonal proliferation, (c) that normally it is transmitted in a regular fashion (with exceptions) to sexual offspring, and (d) that it is inherited as a single dominant trait. Data from DNA hybridisation and enzyme assays confirm this interpretation. The functional gene is integrated together with several non-functional copies and bacterial plasmid sequences, which are inherited as one block together with the functional gene.

Chimeric vector construction for higher-plant transformation

A chimeric vector pKR612B1 was developed containing the neomycin phosphotransferase (APH) gene from the Tn 5 transposon under the control of the gene VI promoter of cauliflower mosaic virus (CaMV), and was used to transform higher plant protoplasts. Plasmid pDOB612, the parental vector of pKR612B1, has two unique restriction sites, SmaI and BamHI, positioned just downstream of the CaMV gene VI promoter sequence. These unique cloning sites can be used for any kind of gene insertion into this vector. Using the polyethylene glycol transformation procedure, a large number of turnip and tobacco protoplasts were transformed and proved to be resistant to kanamycin (Km). From tobacco protoplasts whole Km-resistant plants were regenerated and shown to contain the integrated foreign gene. APH activity was detected in both transformed calli and in regenerated plants. DNA from transformed clones was analysed by Southern blot hybridization, showing the presence of the Tn5-derived gene.

Direct gene transfer to plants

Evidence for direct, gene-mediated stable genetic transformation of plant cells of Nicotiana tabacum is presented. A selectable hybrid gene comprising the protein coding region of the Tn5 aminoglycoside phosphotransferase type II gene under control of cauliflower mosaic virus gene VI expression signals was introduced into plant protoplasts as part of an Escherichia coli plasmid. The gene was stably integrated into plant genomic DNA and constitutively expressed in selected, drug resistant, protoplast-derived cell clones. The mode of integration of the foreign gene into the plant genome resembled that observed for DNA transfection of mammalian cells. Plants regenerated from transformed cell lines were phenotypically normal and fertile, and they maintained and expressed the foreign gene throughout the development of vegetative and generative organs. Microspores, grown in anther culture, developed into resistant and sensitive haploid plantlets. Genetic crossing analysis of one of the transformed plants revealed the presence of one dominant trait for kanamycin resistance segregating in a Mendelian fashion in the F(1) generation.