Isolation and characterization of cDNA clones encoding pathogenesis-related proteins from tobacco mosaic virus infected tobacco plants.

Infection of the tobacco cultivar Samsun NN by tobacco mosaic virus (TMV) results in a hypersensitive response. During this defense reaction several host encoded proteins, known as pathogenesis-related proteins (PR-proteins), are induced. Poly(A)+ RNA from TMV infected tobacco plants was used to construct a cDNA library. Thirty two cDNA clones were isolated and after digestion with different restriction endonucleases, twenty clones were found to code for PR-1a, six clones for PR-1b, and four clones for PR-1c. Two independent cDNA clones of each class were further characterized by DNA sequence analysis. All clones analyzed contained the 138 amino acid coding regions of their respective mature proteins, but only partial sequences of the signal peptides. Minor differences between the nucleotide sequences for clones belonging to the same class were detected. Comparison of the amino acid sequence for PR-1a deduced from its nucleotide sequence with published data obtained by Edman degradation of the protein showed four differences. Analysis of the 3' ends of the cDNA clones indicates that various alternate poly(dA) addition sites are used. Southern blot analysis using these cDNAs as probes suggests the presence of multiple PR-protein genes in the genomes of tobacco and tomato plants.     

Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin-like protein,chitinase and glucanase against Fusarium graminearum

Genes encoding pathogenesis-related (PR-) proteins isolated from a cDNA library of Fusarium graminearum-infected wheat spikes of scab-resistant cultivar 'Sumai-3' were transformed into susceptible spring wheat, 'Bobwhite' using a biolistic transformation protocol, with the goal of enhancing levels of resistance against scab. Twenty-four putative transgenic lines expressing either a single PR-protein gene or combinations thereof were regenerated. Transgene expression in a majority of these lines (20) was completely silenced in the T(1) or T(2) generations. Four transgenic wheat lines showed stable inheritance and expression of either a single transgene or transgene combinations up to four generations. One line co-expressing a chitinase and beta-1,3-glucanase gene combination, when bioassayed against scab showed a delay in the spread of the infection (type II resistance) under greenhouse conditions. This line and a second transgenic line expressing a rice thaumatin-like protein gene (tlp) which had moderate resistance to scab in previous greenhouse trials, along with susceptible and resistance checks were evaluated for resistance to scab under field conditions. None of the transgenic lines had resistance to scab in the field under conditions of strong pathogen, suggesting these plants lacked effective resistance to initial infection (type I resistance) under these conditions. As far as is known, this is the first report of field evaluation of transgenic wheat expressing genes for PR-proteins against disease resistance.     

Purification and characterization of 8 of the pathogenesis-related proteins in tobacco leaves reacting hypersensitively to tobacco mosaic virus

Summary Leaves of tobacco plants (Nicotiana tabacum cv. Samsun NN) which are reacting hypersensitively to infection with tobacco mosaic virus contain 10 major pathogenesis-related (PR) proteins which are absent, or present in small amounts in uninfected leaves. We describe here a preparative procedure of purification of the tobacco PR-proteins which involves a combination of conventional and high-performance liquid chromatography. The separation and isolation of the proteins were based on differences in net charge at different pH values, in isoelectric point and in apparent molecular weight. This procedure led to the purification to homogeneity of 8 PR-proteins, as shown by polyacrylamide slab gel electrophoresis (PAGE) of the purified proteins under denaturing and non-denaturing conditions. These were the 3 well-known proteins PR-1a,-1b and-1c, and 5 other major PR-proteins, called PR-2,-N,-O,-P and-Q, according to the nomenclature of Van Loon (39). None of the purified PR-proteins gave a positive Schiff reaction for carbohydrate content. Molecular weight determinations from gel permeation chromatography and from sodium dodecyl sulphate (SDS)-PAGE indicated that all 8 PR-proteins were monomers and that three groups could be distinguished among them. The first group is the PR-1 group containing PR-1a,-1b and-1c (12000 MW), the second consists of PR-P and PR-Q (14000 MW) and the third of PR-2, PR-N and PR-O (25000 MW). In the PR-1 group, PR-1a can be distinguished clearly from the two other members on denaturing slab gels containing both SDS and urea.     

Virus-induced synthesis of messenger RNAs for precursors of pathogenesis-related proteins in tobacco

Infection of Samsun NN tobacco with tobacco mosaic virus (TMV) induces a number of host-encoded, so-called pathogenesis-related (PR-) proteins, which are found in the intercellular space of the leaf and are associated with induced resistance. By immunoprecipitation of their in vitro translation products we were able to detect the mRNAs corresponding to a number of PR-proteins in TMV-infected tobacco, but not in healthy plants. Analysis by the Northern blot technique using cloned cDNA of PR1-mRNAs as probe showed that the mRNAs for the closely related proteins PR1a, 1b and 1c occur at a low level in healthy tobacco; upon TMV infection this level is increased > 100-fold. The PR1-specific probe did no hybridize to mRNAs corresponding to other PR-proteins. Sequencing of the 5'-terminal region of PR1-mRNAs showed that PR1-proteins are derived from precursors by removal of an N-terminal signal peptide of 30 amino acids.     

Genetic enrichment of cereal crops via alien gene transfer: New challenges

Genetic improvement of crops has traditionally been achieved through sexual hybridization between related species, which has resulted in numerous cultivars with high yields and superior agronomic performance. Conventional plant breeding, sometimes combined with classical cytogenetic techniques, continues to be the main method of cereal crop improvement. More recently, through the introduction of new tools of biotechnology, crossing barriers have been overcome, and genes from unrelated sources have become available to be introduced asexually into plants. Cereal crops were initially difficult to genetically engineer, mainly due to their recalcitrance to in vitro regeneration and their resistance to Agrobacterium infection. Systematic screening of cultivars and explant tissues for regeneration potential, development of various DNA delivery methods and optimization of gene expression cassettes have produced transformation protocols for the major cereals, although some elite cultivars still remain recalcitrant to transformation. Most of the transgenic cereals developed for commercial purpose exhibit herbicide and/or insect resistance; traits that are often controlled by a single gene. In recent years, more complex traits, such as dough functionality in wheat and nutritional quality of rice have been improved by the use of biotechnology. The current challenges for genetic engineering of plants will be to understand and control factors causing transgene silencing, instability and rearrangement, which are often seen in transgenic plants and highly undesirable in lines to be used for crop development. Further improvement of current cereal cultivars is expected to benefit greatly from information emerging from the areas of genomics, proteomics and bioinformatics.     

Molecular genetics of disease resistance in cereals

AIMS: This Botanical Briefing attempts to summarize what is currently known about the molecular bases of disease resistance in cereal species and suggests future research directions. SCOPE: An increasing number of resistance (R) genes have been isolated from rice, maize, wheat and barley that encode both structurally related and unique proteins. This R protein diversity may be attributable to the different modus operandi employed by pathogen species in some cases, but it is also a consequence of multiple defence strategies being employed against phytopathogens. Mutational analysis of barley has identified additional genes required for activation of an R gene-mediated defence response upon pathogen infection. In some instances very closely related barley R proteins require different proteins for defence activation, demonstrating that, within a single plant species, multiple resistance signalling pathways and different resistance strategies have evolved to confer protection against a single pathogen species. Despite the apparent diversity of cereal resistance mechanisms, some of the additional molecules required for R protein function are conserved amongst cereal and dicotyledonous species and even other eukaryotic species. Thus the derivation of functional homologues and interacting partner proteins from other species is contributing to the understanding of resistance signalling in cereals. The potential and limit of utilizing the rice genome sequence for further R gene isolation from cereal species is also considered, as are the new biotechnological possibilities for disease control arising from R gene isolation. CONCLUSIONS: Molecular analyses in cereals have further highlighted the complexity of plant-pathogen co-evolution and have shown that numerous active and passive defence strategies are employed by plants against phytopathogens. Many advances in understanding the molecular basis of disease resistance in cereals have focused on monogenic resistance traits. Future research targets are likely to include less experimentally tractable, durable polygenic resistances and nonhost resistance mechanisms.     

Agrobacterium-mediated transformation of cereals: a promising approach crossing barriers

Cereal crops have been the primary targets for improvement by genetic transformation because of their worldwide importance for human consumption. For a long time, many of these important cereals were difficult to genetically engineer, mainly as a result of their inherent limitations associated with the resistance to Agrobacterium infection and their recalcitrance to in vitro regeneration. The delivery of foreign genes to rice plants via Agrobacterium tumefaciens has now become a routine technique. However, there are still serious handicaps with Agrobacterium -mediated transformation of other major cereals. In this paper, we review the pioneering efforts, existing problems and future prospects of Agrobacterium -mediated genetic transformation of major cereal crops, such as rice, maize, wheat, barley, sorghum and sugarcane.     

The Extracellular Acidic and Basic Pathogenesis-Related Proteins of Soybean Induced by Viral Infection

Seventeen major host-encoded pathogenesis related (PR)-proteins have been found in intercellular fluids of necrotic virus-infected soybean leaves. None of them was present in fluids of healthy controls. By native and SDS-denaturing polyacrylamide gel electrophoresis, ten major acidic PR-proteins have been identified and classified on the basis of their molecular weight in three groups: group 1 included four proteins of 16–17 Kd; group 2, three proteins of 26 Kd; group 3, three proteins of 32 Kd. Seven PR-proteins were basic, and were classified in three groups: group 1 included three proteins of 16 Kd; group 2, one protein of 23 Kd; group 3, three proteins of 32 Kd. As found for tobacco and potato, soybean PR-protein patterns show high number of acidic and basic proteins.     

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.     

Structure and evolution of cereal genomes

The cereal species, of central importance to our diet, began to diverge 50-70 million years ago. For the past few thousand years, these species have undergone largely parallel selection regimes associated with domestication and improvement. The rice genome sequence provides a platform for organizing information about diverse cereals, and together with genetic maps and sequence samples from other cereals is yielding new insights into both the shared and the independent dimensions of cereal evolution. New data and population-based approaches are identifying genes that have been involved in cereal improvement. Reduced-representation sequencing promises to accelerate gene discovery in many large-genome cereals, and to better link the under-explored genomes of 'orphan' cereals with state-of-the-art knowledge.     

植物抗虫基因工程研究进展

植物抗虫基因工程为防治农业害虫提供了一条崭新途径。本文对植物抗虫基因工程近年来所取得的某些研究进展,包括目前已发现和利用的抗虫基因、提高抗虫基因在植物体内表达的方法以及防止或延缓害虫产生抗性的策略等方面进行了综合评述,并对植物抗虫基因工程中有待解决的问题和发展前提提出了自己的看法。     

Pathogenesis-related acidic beta-1,3-glucanase genes of tobacco are regulated by both stress and developmental signals

Three pathogenesis-related (PR) proteins of tobacco are acidic isoforms of beta-1,3-glucanase (PR-2a, -2b, -2c). We have cloned and sequenced a partial cDNA clone (lambda FJ1) corresponding to one of the PR-2 beta-1,3-glucanases. A small gene family encodes the PR-2 proteins in tobacco, and similar genes are present in a number of plant species. We analyzed the stress and developmental regulation of the tobacco PR-2 beta-1,3-glucanases by using northern and western analyses and a new technique to assay enzymatic activity. Stress caused by both thiamine and tobacco mosaic virus (TMV) infection resulted in a dramatic increase in the levels of PR-2 mRNA, protein, and enzyme activities. The increased PR-2 gene expression in upper uninoculated leaves of plants infected with TMV also suggests a role in systemic acquired resistance. During floral development, a number of beta-1,3-glucanase activities were observed in all flower tissues. However, PR-2 polypeptides were observed only in sepal tissue. In contrast, an mRNA that hybridized to the PR-2 cDNA was present in stigma/style tissue and the sepals. Primer extension analysis confirmed the identity of the PR-2 mRNA in sepals, but indicated that the beta-1,3-glucanase gene expressed in the stigma/style of flowers was distinct from the PR-2 genes. The induction of PR-2 protein synthesis by both stress and developmental signals was accompanied by a corresponding increase in the steady-state levels of PR-2 mRNA, suggesting that PR-2 gene expression is regulated, in part, at the level of mRNA accumulation.     

植物抗虫基因工程研究进展

植物抗虫基因工程为防治农业害虫提供了一条崭新途径。本文对植物抗虫基因工程近年来所取得的某些研究进展,包括目前已发现和利用的抗虫基因、提高抗虫基因在植物体内表达的方法以及防止或延缓害虫产生抗性的策略等方面进行了综合评述,并对植物抗虫基因工程中有待解决的问题和发展前景提出了自己的看法。     

抗虫植物基因工程研究进展

虫害是造成农业减产的主要原因之一。据不完全统计,全世界每年因虫害引起的作物减产达总产量的15％,损失高达数千亿美元。在我国,因虫害水稻减产在lO％以上;小麦减产近20％;棉花减产在