Modification of disease resistance of tobacco callus tissues by cytokinins

The effects of differing cytokinin and auxin concentrations on resistance of tobacco (Nicotiana tabacum L.) tissue cultures to race 0 of Phytophthora parasitica var. nicotianae were examined. With 1 micromolar kinetin and either 11.5 micromolar indoleacetic acid or 1 micromolar 2,4-dichlorophen-oxyacetic acid, tissues from resistant cultivars exhibited a “hypersensitive” reaction to zoospores of the fungus and subsequently were colonized only slightly. With susceptible cultivars or with tissues from resistant cultivars supplied with higher cytokinin levels (e.g. 10 micromolar kinetin), this hypersensitive reaction did not occur and tissues were heavily colonized. Benzylaminopurine and kinetin were particularly effective in eliminating both the hypersensitive reaction and disease resistance. Zeatin and 6-(3-methyl-2-butenylamino)purine were less effective. Increases in indoleacetic acid levels reversed the effects of high cytokinin concentrations. The balance of phytohormones apparently controls the host response to the fungus; thus, in this system, resistance or susceptibility can be studied without changing either host or fungal genotype.     

In vitro replication of tobacco mosaic virus RNA in tobacco callus cultures: solubilization of membrane-bound replicase and partial purification.

A fraction containing membrane-bound tobacco mosaic virus RNA replicase was isolated form tobacco mosaic virus-infected tobacco callus cultures. The replicase activity reached a maximum 60 h after inoculation and then declined. The enzyme activity was insensitive to actinomycin D and DNase. The corresponding fraction from healthy callus contained essentially no activity. The viral RNA synthesis in vitro proceeded linearly for 30 min and required the four nucleotide triphosphates and Mg2+ ions. Mn2+ was a poor substitute for Mg2+. During RNA synthesis the product was at least 70% resistant to RNase in 2X SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digested by RNase in 0.1X SSC. Analysis of the product by polns) that appeared to be replicative form and a partially RNase-resistant structure similar to replicative intermediate form. Washing the membrane-bound replicase with Mg2+-deficient buffer solubilized enzyme. The solubulized enzyme was further purified by DEAE-Sephadex column chromatography. The DEAE-purified enzyme was nearly completely dependent upon tobacco mosaic virus RNA for activity. Analysis of the product on a sucrose gradient revealed a double-stranded RNA with sedimentation of 16S and smaller heterogeneous RNase-sensitive products.     

Changes in soluble proteins in callus cells of hypersensitive tobacco inoculated with tobacco mosaic virus

Summary Protein changes occurred in callus cells of hypersensitive tobacco (Nicotiana tabacum var. Xanthi-nc) 72 hr after inoculation with tobacco mosaic virus and incubation on a minimal growth medium. Two protein bands, serologically related to viral coat protein, were obtained from extracts of infected cells following electrophoresis on 7% and 10% polyacrylamide gels. An additional, slower migrating protein, perhaps due to virus-induced stimulation of a host protein, also was detected. Although local lesions appeared on callus after 40 hr of incubation, four proteins previously reported in lesion-bearing hypersensitive tobacco leaves were not found. The possible significance of this and the usefulness of a callus-TMV system as a tool to study virus-induced protein changes are discussed. Michigan Agricultural Experiment Station Journal Paper No. 7191.     

烟草花叶病毒复制酶介导抗性的研究进展

在抗病毒植物基因工程中,利用病毒的复制酶基因是一种很有前途的方法。本对烟草花叶病毒（TMV）的基因组结构及其编码的蛋白的功能作了简介,同时较详细地阐述了由TMV复制本科的通读部分、全长复制酶以及突变或缺失的复制酶介导的对病毒抗性的研究进展。     

Reactions and resistance ofChenopodium species to tobacco mosaic virus

Chenopodium species react on infection with tobacco mosaic virus by the formation of chlorotic or necrotic lesions and later by the abscission of infected leaves. A transition of local infection into the stem has been observed exceptionally inChenopodium quinoa, C. hybridum, andC. rubrum, but no systemic infection of the leaves followed. Systemic infection was demonstrated only inC. polyspermum andC. murale. The recovery of new sprouts was demonstrated in C.murale in the late chronic phase of infection.     

Polyacrylic acid-induced resistance to tobacco mosaic virus in tobacco cv. Xanthi

Polyacrylic acid (PA) of molecular weight 1700A, 1700B and 3500 caused resistance to infection with tobacco mosaic virus in tobacco cv. Xanthi-nc, when sprayed on the leaves or watered into the soil. The numbers of lesions produced in the treated plants were between 27 and 97% fewer than in the untreated plants depending on the concentration of PA, its molecular weight and the method of application. Some resistance was caused against potato virus X and potato virus Y but only at concentrations that were harmful to the plants. It appears that PA activates a mechanism responsible for localizing viruses in hypersensitive plants.     

Dark and light green tissues of tobacco leaves systemically infected with tobacco mosaic virus

There are significant changes in the structure of the upper tobacco (Nicotiana tabacum L.) leaves systemically infected with tobacco mosaic virus (TMV) especially in the light green tissue (LGT). Dark green areas (DGI) had intermediate status between healthy tissue and LGT. DGI contained significantly less infectious TMV and viral antigen than the LGT. The DGI, LGT and healthy tissues did not differ in the permeability of cell membranes and in the set of acidic pathogenesis-related (PR) proteins but the total content of PR-proteins in the healthy plants was higher than in the infected ones with the DGI being intermediate between healthy tissue and LGT. The crude leaf extracts from DGI and LGT showed less total ribonuclease activity and ribonuclease isozymes in comparison with control.     

Antimicrobial peptaibols induce defense responses and systemic resistance in tobacco against tobacco mosaic virus

Trichoderma spp. are well-known biocontrol agents because of their antimicrobial activity against bacterial and fungal phytopathogens. However, the biochemical mechanism of their antiviral activity remains largely unknown. In this study, we found that Trichokonins, antimicrobial peptaibols isolated from Trichoderma pseudokoningii SMF2, could induce defense responses and systemic resistance in tobacco (Nicotiana tabacum var. Samsun NN) against tobacco mosaic virus (TMV) infection. Local Trichokonin (100 nM) treatment led to 54% lesion inhibition, 57% reduction in average lesion diameter and 30% reduction in average lesion area in systemic tissue of tobacco compared with control, indicating that Trichokonins induced resistance in tobacco against TMV infection. Trichokonin treatment increased the production of reactive oxygen species and phenolic compounds in tobacco. Additionally, application of Trichokonins significantly increased activities of pathogenesis-related enzymes PAL and POD, and upregulated the expression of several plant defense genes. These results suggested that multiple defense pathways in tobacco were involved in Trichokonin-mediated TMV resistance. We report on the antivirus mechanism of peptaibols, which sheds light on the potential of peptaibols in plant viral disease control.     

An analysis of tobacco mosaic virus replicative structures synthesized in vitro

Summary The RNA structures synthesized in vitro by a crude enzyme complex from tobacco mosaic virus (TMV)-infected leaves have been analyzed; the major viral-specific products were similar to TMV-replicative form (RF) and-replicative intermediate (RI) in electrophoretic behavior and ribonuclease sensitivity. Synthesis of these RF-like and RI-like structures neither required nor responded to added viral RNA, but did require all four ribonucleotide triphosphates. Enriched radiolabeled RF-like and RI-like RNA fractions were isolated from non-denaturing agarose gels by electroelution and hybridized to a collection of TMV sequences cloned into bacteriophage M13. Enriched RF-RNA hybridized to sequences of both plus and minus polarity, while enriched RI-RNA hybridized only to inserts of minus polarity, indicating only plus strand synthesis in this fraction. Most of the label incorporated into the plus strand of the enriched RF-RNA was found near the 3-end of this strand, while most of the label incorporated into enriched RI-RNA was found several hundred bases from the 5-end of the plus strand.Paper presented at the first International Congress of Plant Molecular Biology (Savannah, GA, 1985).     

The isolation of tobacco mosaic virus RNA fragments containing the origin for viral assembly

Upon mixing purified TMV RNA with limited amounts of viral coat protein in the form of the disk aggregate, a unique region of the whole RNA becomes protected from nuclease digestion. The protected RNA consists of fragments up to 500 nucleotides long in varying yields, which are found in nucleoprotein particles having a protein-nucleic acid ratio similar to the mature virus. The protected RNA, when reextracted, is able to rebind to coat protein disks rapidly, quantitatively and with high affinity, becoming once more RNAase-resistant in the process. Small aggregates of TMV protein (A protein) are inactive in formation of the nuclease-resistant complexes. On the basis of this evidence, we identify the isolated RNA fragments as portions of TMV RNA containing the origin or initiation site for in vitro reassembly, which have been protected from digestion by incorporation into assembly nucleation complexes.The yield, but not the length distribution, of the protected RNA pieces is found to double upon increasing the protein added from 1–2 disk-equivalents of protein per RNA molecule. This implies that the formation of the nucleation complexes may involve a highly cooperative initial addition of protein.     

Coat-protein-mediated resistance to tobacco mosaic virus: discovery mechanisms and exploitation

In 1986 we reported that transgenic plants which accumulate the coat protein of tobacco mosaic virus (TMV) are protected from infection by TMV, and by closely related tobamoviruses. The phenomenon is referred to as coat-protein-mediated resistance (CP-MR), and bears certain similarities to cross protection, a phenomenon described by plant pathologists early in this century. Our studies of CP-MR against TMV have demonstrated that transgenically expressed CP interferes with disassembly of TMV particles in the inoculated transgenic cell. However, there is little resistance to local, cell-to-cell spread of infection. CP-MR involves interaction between the transgenic CP and the CP of the challenge virus, and resistance to TMV is greater than to tobamo viruses that have CP genes more distantly related to the transgene. Using the known coordinates of the three-dimensional structure of TMV we developed mutant forms of CP that have stronger inter-subunit interactions, and confer increased levels of CP-MR compared with wild-type CP. Similarly, it is predicted that understanding the cellular and structural basis of CP-MR will lead to the development of variant CP transgenes that each can confer high levels of resistance against a range of tobamoviruses.