John F. Bol

Differential induction of acquired resistance and PR gene expression in tobacco by virus infection, ethephon treatment, UV light and wounding

Genes for acidic, extracellular and basic, intracellular pathogenesis-related (PR) proteins of tobacco were studied for their response to tobacco mosaic virus (TMV) infection, ethephon treatment, wounding and UV light. The genes encoding the acidic PR proteins (PR-1, PR-2, PR-3, PR-4 and PR-5) responded similarly to the different forms of stress. They appeared to be highly inducible by TMV, moderately inducible by ethephon treatment and UV light and not inducible by wounding. The genes for the basic counterparts of PR-1, PR-2, PR-3 and PR-5 also displayed a common stress response. However, this response was different from that of the acidic PR proteins. Here, the highest induction was obtained upon ethephon treatment, while the other stress conditions resulted in somewhat lower levels of expression. Most genes for acidic PR proteins are systemically induced in the uninfected upper leaves of TMV-infected plants, whereas the genes encoding the basic PR proteins are not. Increased levels of resistance to TMV, comparable to resistance obtained by pre-infection with the virus, were found in UV-irradiated leaves but not in wounded or ethephon-treated leaves. This indicates that the basic PR proteins are not involved in resistance to TMV infection. Tobacco phenylalanine ammonia-lyase genes were not inducible by the various stress conditions. The implications of these findings in relation to the phenomenon of acquired resistance are discussed.

Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance.

After a hypersensitive response to invading pathogens, plants show elevated accumulation of salicylic acid (SA), induced expression of plant defense genes, and systemic acquired resistance (SAR) to further infection by a broad range of pathogens. There is compelling evidence that SA plays a crucial role in triggering SAR. We have transformed tobacco with two bacterial genes coding for enzymes that convert chorismate into SA by a two-step process. When the two enzymes were targeted to the chloroplasts, the transgenic (CSA, constitutive SA biosynthesis) plants showed a 500- to 1,000-fold increased accumulation of SA and SA glucoside compared to control plants. Defense genes, particularly those encoding acidic pathogenesis-related (PR) proteins, were constitutively expressed in CSA plants. This expression did not affect the plant phenotype, but the CSA plants showed a resistance to viral and fungal infection resembling SAR in nontransgenic plants.

A functional equivalence of top component a RNA and coat protein in the initiation of infection by alfalfa mosaic virus

Abstract The nucleoproteins of alfalfa mosaic virus (AMV) were purified by zonal gradient centrifugation, and their homogeneity and RNA contents were established by gel electrophoresis. Each component was shown to contain one type of RNA of a specific length. Combinations of purified preparations were infectious only when the bottom component (B), middle component (M), and the top component b (Tb) nucleoproteins were present. However, no infectivity was found in a mixture of the three corresponding RNAs. Infection could be started by adding to this mixture a fourth RNA, the top component a RNA (Ta-RNA). The fact that a mixture of B, M, and Tb nucleoprotein is infectious while the corresponding nucleic acid mixture is not, is explained by the finding that the latter becomes infectious upon addition of low amounts of AMV coat protein. Evidence is presented that the activity of AMV coat protein is not due to protection of the RNAs against degrading enzymes. The function of Ta-RNA and its translation product—the coat protein—in the onset of infection is discussed.

Identification of potyviruses using the polymerase chain reaction with degenerate primers

Local areas of conserved amino acid sequence in the replicase and coat proteins of potyviruses were used to select nucleotide sequences for use in the construction of sets of degenerate oligonucleotide primers for amplification of DNA fragments on potyvirus-specific templates in a combined assay of reverse transcription and the polymerase chain reaction (RT-PCR). Sequences selected for the construction of degenerate primers included the coat protein gene sequence of tulip breaking virus from lily, which is reported in this paper. It is shown that the degenerate primers support potyvirus-specific amplification, but do not support amplification on carlavirus and potexvirus templates. A panel consisting of definite and prospective members of the potyvirus group occurring in bulbous crops was subjected to the degenerate primer RT-PCR assay; amplified fragments were used in cross-hybridization experiments and restriction fragment length polymorphism analysis to detect relationships among these potyviruses. A partially characterized virus isolated from Gloriosa rothschildiana was positively identified as a potyvirus by specific amplification and subsequent sequence analysis of an amplified DNA fragment.

Systemic Resistance in Arabidopsis Induced by Rhizobacteria Requires Ethylene-Dependent Signaling at the Site of Application

Root colonization of Arabidopsis thaliana by the nonpathogenic, rhizosphere-colonizing, biocontrol bacterium Pseudomonas fluorescens WCS417r has been shown to elicit induced systemic resistance (ISR) against Pseudomonas syringae pv. tomato (Pst). The ISR response differs from the pathogen-inducible systemic acquired resistance (SAR) response in that ISR is independent of salicylic acid and not associated with pathogenesis-related proteins. Several ethylene-response mutants were tested and showed essentially normal symptoms of Pst infection. ISR was abolished in the ethylene-insensitive mutant etr1-1, whereas SAR was unaffected. Similar results were obtained with the ethylene-insensitive mutants ein2 through ein7, indicating that the expression of ISR requires the complete signal-transduction pathway of ethylene known so far. The induction of ISR by WCS417r was not accompanied by increased ethylene production in roots or leaves, nor by increases in the expression of the genes encoding the ethylene biosynthetic enzymes 1-aminocyclopropane-1-carboxylic (ACC) synthase and ACC oxidase. The eir1 mutant, displaying ethylene insensitivity in the roots only, did not express ISR upon application of WCS417r to the roots, but did exhibit ISR when the inducing bacteria were infiltrated into the leaves. These results demonstrate that, for the induction of ISR, ethylene responsiveness is required at the site of application of inducing rhizobacteria.

A conformational switch at the 3′ end of a plant virus RNA regulates viral replication

3′ untranslated regions of alfamo‐ and ilar‐virus RNAs fold into a series of stem–loop structures to which the coat protein binds with high affinity. This binding plays a role in initiation of infection (‘genome activation’) and has been thought to substitute for a tRNA‐like structure that is found at the 3′ termini of related plant viruses. We propose the existence of an alternative conformation of the 3′ ends of alfamo‐ and ilar‐virus RNAs, including a pseudoknot. Based on (i) phylogenetic comparisons, (ii) in vivo and in vitro functional analyses of mutants in which the pseudoknot has been disrupted or restored by compensatory mutations, (iii) competition experiments between coat protein and viral replicase, and (iv) investigation of the effect of magnesium, we demonstrate that this pseudoknot is required for replication of alfalfa mosaic virus. This conformation resembles the tRNA‐like structure of the related bromo‐ and cucumo‐viruses. A low but specific interaction with yeast CCA‐adding enzyme was found. The existence of two mutually exclusive conformations for the 3′ termini of alfamo‐ and ilar‐virus RNAs could enable the virus to switch from translation to replication and vice versa. The role of coat protein in this modulation and in genome activation is discussed.

Expression of alfalfa mosaic virus and tobacco rattle virus coat protein genes in transgenic tobacco plants

Using the Agrobacterium tumefaciens binary vector system, a chimeric gene consisting of the cauliflower mosaic virus 35 S promoter, alfalfa mosaic virus (AIMV) coat protein (CP) cistron, and the nopaline synthase polyadenylation signal was integrated into the genome of Nicotiana tabacum cv. Samsun NN. In 70% of the transgenic tobacco plants the chimeric mRNA and its translation product could be detected. CP accumulated to levels up to 0.05% of the soluble leaf protein. The accumulation was independent of leaf age. The same approach was undertaken for the CP of tobacco rattle virus (TRV). The chimeric gene was integrated in the genome of Nicotiana tabacum cv. Xanthi nc. The results with respect to the accumulation of the chimeric mRNA and TRV CP in leaves of transgenic tobacco plants were comparable to those with AIMV transformed plants. Plants accumulating AIMV CP were highly resistant to infection with AIMV nucleoproteins but could be infected with a mixture of AIMV RNAs 1-4. Moreover, a mixture of AIMV RNAs 1, 2, and 3 was infectious to these plants but not to nontransformed control plants.

Molecular characterization of messenger RNAs for 'pathogenesis-related' proteins 1a, 1b and 1c, induced by TMV infection of tobacco.

A cDNA library was made to poly(A)‐containing RNA from tobacco mosaic virus (TMV)‐infected Samsun NN tobacco plants and clones corresponding to mRNAs for the ‘pathogenesis‐related’ (PR) proteins 1a, 1b and 1c were identified. One clone was found to contain a complete copy of PR‐1b mRNA. The structural organization of this RNA is: a leader sequence of 29 nucleotides, an open reading frame of 504 nucleotides encoding a 30 amino acid long signal peptide and a 138 amino acid long mature protein, and a 3′‐non‐coding region of 235 nucleotides. Two other clones were found to contain partial copies of PR‐1a and PR‐1c mRNAs. The data indicate an ∼90% homology between the amino acid sequences of PR‐1a, ‐1b and ‐1c. Using one of the clones as probe it was shown that in the TMV‐inoculated lower leaves and the non‐inoculated upper leaves of a tobacco plant, the PR‐1 mRNAs become detectable from 2 and 8 days after inoculation, respectively.

A Novel WRKY Transcription Factor Is Required for Induction of PR-1a Gene Expression by Salicylic Acid and Bacterial Elicitors

PR-1a is a salicylic acid-inducible defense gene of tobacco (Nicotiana tabacum). One-hybrid screens identified a novel tobacco WRKY transcription factor (NtWRKY12) with specific binding sites in the PR-1a promoter at positions −564 (box WK1) and −859 (box WK2). NtWRKY12 belongs to the class of transcription factors in which the WRKY sequence is followed by a GKK rather than a GQK sequence. The binding sequence of NtWRKY12 (WK box TTTTCCAC) deviated significantly from the consensus sequence (W box TTGAC[C/T]) shown to be recognized by WRKY factors with the GQK sequence. Mutation of the GKK sequence in NtWRKY12 into GQK or GEK abolished binding to the WK box. The WK1 box is in close proximity to binding sites in the PR-1a promoter for transcription factors TGA1a (as-1 box) and Myb1 (MBSII box). Expression studies with PR-1a promoter∷β-glucuronidase (GUS) genes in stably and transiently transformed tobacco indicated that NtWRKY12 and TGA1a act synergistically in PR-1a expression induced by salicylic acid and bacterial elicitors. Cotransfection of Arabidopsis thaliana protoplasts with 35S∷NtWRKY12 and PR-1a∷GUS promoter fusions showed that overexpression of NtWRKY12 resulted in a strong increase in GUS expression, which required functional WK boxes in the PR-1a promoter.

Alfalfa mosaic virus and ilarviruses: involvement of coat protein in multiple steps of the replication cycle

IP: 54.70.40.11 On: Sat, 02 Mar 2019 22:33:49 Journal of General Virology (1999), 80, 1089–1102. Printed in Great Britain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .