Raymond F. White

Isolation and characterization of a cDNA clone encoding the anti-viral protein from Phytolacca americana

Phytolacca anti-viral protein (PAP) was purified from Phytolacca leaves and the N-terminal was sequenced. A cDNA library was made from mRNAs isolated from Phytolacca leaves and cDNA clones for PAP were identified using oligonucleotide probes derived from the N-terminal amino acid sequence. The PAP-cDNA clone was sequenced from both directions. The predicted amino acid sequence of PAP was compared with the amino acid sequences of other ribosome-inactivating proteins. The identities of these proteins to PAP ranged from 29 to 38%, and a region was found in each with a sequence similar to the PAP sequence (AIQMVSEAARFKYI). Southern blot analysis indicates that PAP is encoded by a multi-gene family.

Changes with time, in the distribution of virus and PR protein around single local lesions of TMV infected tobacco

SummaryELISA was used to determine PR la protein and TMV accumulation in local necrotic lesions produced on salicylic acid and water sprayed Nicotiana tabacum cv Xanthi-nc leaves. The amount of PR la protein produced is the result of an interaction between the salicylic acid treatment and lesion growth. The implication of these observations for experiments investigating the relationship between PR proteins and resistance are discussed.The distribution of TMV and PR la protein in and around single local necrotic lesions up to 14 days after inoculation was measured by ELISA. The highest concentration of TMV was in the centre of the lesion and decreased rapidly with distance from the centre. In contrast there was very little PR la protein in the centre of the lesion, the largest amounts were just outside the centre, and the concentration then decreased with distance from the centre. This is the distribution that might be expected for a substance closely associated with the restriction of virus spread.

The detection of PR (b) protein and TMV by ELISA in systemic and localised virus infections of tobacco.

An antiserum was prepared to the b1 protein purified from TMV infectedN. tabacum cv. Xanthi-nc leaves and used to study PR proteins. The Xanthi-nc proteins b2 and b3 were shown to be serologically closely related to b1. Antisera to b1 protein and TMV were used in a F(ab′)2 enzyme linked immunosorbent assay to monitor PR protein and TMV concentrations, respectively, during the first 6 days of a systemic TMV infection (cv. Xanthi) and a localised TMV infection (cv. Xanthi-nc).

Virus-induced resistance responses in plants

Abstract Infection of plants by viruses can lead to a systemic or a localized infection. Systemic infections can produce protection against related viruses, as seen in the phenomenon of cross‐protection. The mechanisms thought to be involved are discussed. Localized infections also produce protection but against a wide range of unrelated pathogens, including bacteria and fungi. This involves an active response by the infected plants, switching on a number of different resistance mechanisms for the different types of pathogens. Research on these plant‐coded, induced proteins thought to be involved in resistance is discussed.

Nucleotide sequence of beet cryptic virus 3 dsRNA2 which encodes a putative RNA-dependent RNA polymerase

The nucleotide sequence of a DNA copy of beet cryptic virus 3 double-stranded RNA2 was determined, and one strand was found to contain a single long open reading frame of 1431 nucleotides which encoded a putative polypeptide containing 478 amino acid residues with an M r of 54·9K. This polypeptide contained conserved amino acid sequence motifs found in the genes that encode putative RNA-dependent RNA polymerases of other RNA viruses.

Biochemical and serological characterization of b-proteins fromNicotiana species

SummaryProteins associated with the hypersensitive response (b-proteins) were purified from variousNicotiana species and compared biochemically and serologically. The method developed to purify proteins b1, b2 and b3 ofN. tabacum cv. Xanthi-nc was used to purify b-proteins present inN. sylvestris (b0, b1 and b3) andN. tomentosiformis (b2), the parental species ofN. tabacum, and b1″ from bothN. glutinosa andN. debneyi. Ultracentrifugation and amino acid analysis of some of these proteins has shown that they are very similar and that they are all monomers in their native form (mol wt = 15 700 for b0, b1, b2 and b3; mol wt = 13 800 for b1″).Based on their reactions to an antiserum produced against protein b1 ofN. tabacum cv. Xanthi-nc, 3 serological groups can be recognized which are independent of the source species (I) b0 and b1, (II) b1″ and b2, (III) b3. Thus, proteins in the same serological group but from different species are more closely related than the b-proteins in different serological groups but present in the same species. The implication of this site on the possible phylogeny of b-proteins is discussed.Serological tests confirmed the b-protein present as a constitutive component in the virus resistant interspecific hybrids ofN. glutinosa ×N. debneyi as protein b1″.

Induction and characterization of some of the pathogenesis-related proteins in sugar beet

Abstract Analysis of the intercellular leaf proteins of tobacco necrosis virus (TNV)-infected sugar beet revealed 12 protein bands (B.v. D1–D12) in polyacrylamide gels (Davis system) normally used to separate acidic proteins, and eight protein bands (B.v. R1–R8) in gels (Reisfeld system) used to separate basic proteins. These proteins were either absent from healthy leaves or present in much smaller amounts. Both qualitative and quantitative differences were found in the proteins induced in cv. Sharpes Klein E and in a subline of a Maribo diploid multigerm family. All of these proteins are resistant to degradation by trypsin and chymotrypsin. All but D1, D6 and D7 were soluble at pH 2·8, and all but D1, D3, D4, D9 and D11 were induced by salicylic acid. There were no differences in the number and quantity of intercellular proteins from leaves of healthy plants and those from uninoculated uninfected leaves of TNV-infected plants. Purification of two of the acidic proteins (D2 and D3) was achieved by blotting them onto, and eluting them from, Immobilon. Subsequent analysis in a SDS polyacrylamide gel showed that both D2 and D3 had an apparent molecular weight of 28 kD. Amino acid analysis and partial sequencing of their N-termini showed an 80% homology to induced chitinases from cucumber and Arabidopsis thaliana and a lysozyme from Parthenocissus quinquifolia .

Immunocytochemical Location of Pathogenesis-related b1 Protein Induced in Tobacco Mosaic Virus-infected or Polyacrylic Acid-treated Tobacco Plants

Summary Pathogenesis-related b1 (PR-b1) protein and other serologically related PR-b proteins, known to be associated with both tobacco mosaic virus infection and chemical treatments, such as with polyacrylic acid (PAA), were detected by immunofluorescence microscopy around epidermal, palisade and lacunar leaf cells in hypersensitive tobacco plants (PAA+ line). After gentle fixation and embedding in Lowicryl K4M, PR-b proteins revealed by immunogold labelling were located in the cytoplasm and apoplast. After fixation and embedding in Epon, the PR-b proteins were found to accumulate mainly in the intercellular spaces. These observations support results obtained from biochemical tests.