The role of enzymatic activities of antiviral proteins from plants for action against plant pathogens.

Antiviral proteins (AVPs) from plants possess multiple activities, such as N-glycosidase, RNase, DNase enzymatic activity, and induce pathogenesis-related proteins, salicylic acid, superoxide dismutase, peroxidase, and catalase. The N-glycosidase activity releases the adenine residues from sarcin/ricin (S/R) loop of large subunit of ribosomes and interfere the host protein synthesis process and this activity has been attributed for antiviral activity in plant. It has been shown that AVP binds directly to viral genome-linked protein of plant viruses and interfere with protein synthesis of virus. AVPs also possess the RNase and DNase like activity and may be targeting nucleic acid of viruses directly. Recently, the antifungal, antibacterial, and antiinsect properties of AVPs have also been demonstrated. Gene encoding for AVPs has been used for the development of transgenic resistant crops to a broad range of plant pathogens and insect pests. However, the cytotoxicity has been observed in transgenic crops using AVP gene in some cases which can be a limiting factor for its application in agriculture. In this review, we have reviewed various aspects of AVPs particularly their characteristics, possible mode of action and application.

Purification, characterization and cloning of antiviral/ribosome inactivating protein from Amaranthus tricolor leaves.

An antiviral protein (AVP), imparting high level of resistance against sunnhemp rosette virus (SRV) was purified from the dried leaves of Amaranthus tricolor. The purified protein (AAP-27) exhibited approximately 98% inhibition of local lesion formation at a concentration range of approximately 30 microg ml(-1). The protein was found to be highly basic glycoprotein monomer (pI approximately 9.8) of Mr 27 kDa, with neutral sugar content of 4%. The purified protein exhibited N-glycosidase and RNase activities. We have also isolated full-length cDNA clone, encoding this protein designated as A. tricolor antiviral protein-1 (AAP-1). Two primers, one designed on the basis of N-terminal sequence of the purified protein and the other from the conserved active peptides of other AVPs/RIPs were used for PCR amplification of double stranded cDNA, isolated from the leaves of A. tricolor. The amplified fragment was used as a probe for library screening. The isolated full-length cDNA consisted of 1058 nucleotides with an open reading frame encoding a polypeptide of 297 amino acids. The deduced amino acid sequence of AAP-1 has a putative active domain conserved in other AVPs/RIPs and shows varying homology to the RIPs from other plant species.

RNase and DNase activities of antiviral proteins from leaves of Bougainvillea xbuttiana.

Antiviral proteins (AVPs) purified from the leaves of Bougainvillea xbuttiana cv Mahara exhibited RNase activity against viral RNA of the tobamoviruses, Tobacco mosaic virus (TMV) and Sunnhemp rosette virus (SRV). They caused complete degradation of viral RNAs in a concentration-dependent manner. RNase activity gel assay ruled out the possibility of the presence of contaminating nucleases. AVPs also showed DNase activity, as indicated by conversion of supercoiled form of plasmid DNA into relaxed and linear forms. The implications of these activities in controlling plant viruses are discussed.

Expression of Rhizobium meliloti symbiotic promoters in Azorhizobium caulinodans.

Plasmids containing Rhizobium meliloti symbiotic promoters P1 (promoter of nifHDK) and P2 (promoter of fixABCX) when mobilized into the cells of Azorhizobium caulinodans strain IRBG 46 showed strong expression of these promoters under free-living microaerobic as well as symbiotic conditions. Under free-living conditions microaerobiosis (3% or less O2) was found to be sufficient to activate these promoters; expression being higher at 1% than at 3% O2 concentration. Under symbiotic conditions the expression was much more stronger-with bacteroids in stem nodules showing higher expression than those in root nodules. Under both the conditions expression of the promoters in the native R. meliloti strain Rm102F34 was lower than that in the A. caulinodans strain IRBG 46. The results suggest a functional homology of these promoters in the heterologous background of A. caulinodans.

A systemic resistance inducing antiviral protein with N-glycosidase activity from Bougainvillea xbuttiana leaves.

An antiviral protein from Bougainvillea xbuttiana leaves induced systemic resistance in host plants N. glutinosa and Cyamopsis tetragonoloba against TMV and SRV, respectively which was reversed by actinomycin D, when applied immediately or shortly after antiviral protein treatment. When the inhibitor was applied to the host plant leaves post inoculation, it was effective if applied upto 4 h after virus infection. It also delayed the expression of symptoms in systemic hosts of TMV. The inhibitor showed characteristic N-glycosidase activity on 25S rRNA of tobacco ribosomes, suggesting that it could also be interfering with virus multiplication through ribosome-inactivation process.

Effect of combined nitrogen on the expression of nitrate reductase and nitrite reductase in Azorhizobium caulinodans.

In aerobically grown Azorhizobium caulinodans strain IRBG 46, in vivo expression of nitrate reductase (NR) and nitrite reductase (NiR) requires the presence of either nitrate or nitrite. On the contrary mere microaerobic conditions are sufficient for the expression of NR and NiR, however, addition of nitrate to the growth medium enhanced the activities of the enzymes. Optimum concentration of nitrate for maximum expression of NR and NiR activities was different in aerobic and microaerobic conditions. Nitrite was released into the medium both in aerobic and microaerobic conditions beyond a particular concentration of nitrate in the medium. Dissimilatory nitrate reduction was affected to a lesser extent by ammonium compared to assimilatory nitrate reduction.

Effect of nitrate on nitrogen fixation and nitrate assimilation in Azorhizobium-Sesbania rostrata symbiotic system.

Root nodule formation was inhibited by 30% and 50% respectively at low concentration of 1 mM and 2 mM nitrate, while stem nodule formation was enhanced by 50% only at 1 mM nitrate. The nodule specific nitrogenase activity decreased with the increasing concentration of nitrate. At 1 mM nitrate nitrogenase activity per plant stem nodule was not affected, but it was less than 50% in the root nodules as compared to control. Increasing concentration of nitrate increased in vivo activity of nitrate reductase (NR) significantly in stem, root nodules and leaves. Nodule cytosolic NR utilized both NADH and succinate as electron donor, but not reduced MV. However bacteroidal NR utilised reduced MV as reductant more efficiently than succinate.

Possible mechanism of action of antiviral proteins from the leaves of Chenopodium album L.

Antiviral proteins (AVPs) named CAP-I and CAP-II purified from the leaves of Chenopodium album cv Pusa Bathua-1 induced systemic resistance against tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) in both hypersensitive as well as systemic hosts. An increased accumulation of two polypeptides (approximately 17 kDa and approximately 26 kDa) was observed in untreated upper leaves of Cyamopsis tetragonoloba plants whose basal leaves were treated with CAP-I/CAP-II. Both AVPs exhibited ribosomal RNA N-glycosidase activity on 28S rRNA of tobacco leaves and also caused in vitro degradation of TMV RNA. It is suggested that the CAP-I and -II are multi-functional and may be acting at multiple levels to ensure maximum possible inhibition of viral infection.

Purification and properties of antiviral proteins from the leaves of Bougainvillea xbuttiana.

A non-phytotoxic, resistance inducing, proteinaceous antiviral principle was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration from the leaves of Bougainvillea xbuttiana. It imparted resistance against tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) in their respective test hosts viz. Nicotiana glutinosa, N. tabacum var. Samsun NN, and Cyamopsis tetragonoloba, respectively. The purified principle eluted as a single peak upon gel filtration, but exhibited two polypeptides on SDS-PAGE with Mr 28,000 and 24,000. The two polypeptides were found to be highly basic, rich in lysine with pI around 10.0 and 10.5, respectively. Since this principle effected local lesion inhibition in both treated and untreated top leaves of test host, it might be acting in the initial stages of virus infection as a systemic inducer.

Ribonuclease, deoxyribonuclease, and antiviral activity of Escherichia coli-expressed Bougainvillea xbuttiana antiviral protein 1.

A full-length cDNA encoding ribosome-inactivating/antiviral protein from the leaves of Bougainvillea xbuttiana was recently isolated. The coding region of cDNA was cloned and expressed in Escherichia coli, and the protein product was designated as BBAP1 (Bougainvillea xbuttiana antiviral protein 1). BBAP1 showed ribonuclease activity against Torula yeast RNA. It also exhibited depurination activity against supercoiled pBlueScript SK+ plasmid DNA in a concentration dependent manner, and was found to convert nicked circular DNA into linear form only at higher concentration. On bioassay, BBAP1 exhibited antiviral activity against sunnhemp rosette virus infecting Cyamopsis tetragonoloba leaves in which 95% inhibition of local lesion formation was observed.

An antiviral protein having deoxyribonuclease and ribonuclease activity from leaves of the post-flowering stage of Celosia cristata.

An antiviral protein named CCP-27 was purified from the leaves of Celosia cristata at the post-flowering stage by anion-exchange, cation-exchange, and gel-filtration chromatography. It exhibited resistance against sunnhemp rosette virus in its test host Cyamopsis tetragonoloba. It also exhibited deoxyribonuclease activity against supercoiled pBlueScript SK+ plasmid DNA. It was found to nick supercoiled DNA into nicked circular form at lower protein concentration followed by nicked to linear form conversion at higher protein concentration. CCP-27 also possesses strong ribonuclease activity against Torula yeast rRNA.

Cloning and expression of small cDNA fragment encoding strong antiviral peptide from Celosia cristata in Escherichia coli.

A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714.

Integration ofhup cosmid pHU52 into the chromosomal DNA ofCicer-Rhizobium using Tn5 as an homologous sequence.

Cosmid pHU52, which carrieshup genes ofBradyrhizobium japonicum, has been integrated into theCicer-Rhizobium G36-84 genome via Tn5-mediated homologous recombination. Tn5 was inserted into both the cosmid pHU52 and the chromosome ofCicer-Rhizobium to provide a region of DNA homology, without affecting the expression of necessary genes. An incompatible plasmid, pPH1JI, was used to select those few cells that had undergone recombination. The integration of the cosmid was demonstrated by Southern blot analysis. Chromosomal integration of thehup genes maximized stability and minimized the potential for their horizontal transfer to other bacterial species. The integratedhup genes were found to expressex planta as well in nodules. The method described illustrates how a given gene can be stably integrated into the chromosome.

Cloning of acyl-acyl carrier protein (ACP) thioesterase gene from Brassica juncea.

Acyl-acyl carrier protein (ACP) thioesterase is the chain-length-determining enzyme in de novo biosynthesis of plant fatty acids. For cloning the gene encoding acyl-ACP thioesterase from Brassica juncea, genomic DNA was used as a template to amplify a 0.7 kb thioesterase fragment in a PCR with the primers designed from the known sequences available in the GenBank. This 0.7 kb fragment was used as a probe to study the expression of the gene in developing seeds and also to screen a genomic library of B. juncea constructed in lambdaEMBL-3 to get the full length of the gene. A 4.0 kb BamHI fragment containing the full gene was finally cloned in a plasmid vector from a recombinant phage clone lambda5.12 after a series of screening, sub-cloning and Southern hybridization.