Antibody-Based Resistance to Plant Pathogens

Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments.     

Engineering bacteriocin‐mediated resistance against the plant pathogen Pseudomonas syringae

The plant pathogen, Pseudomonas syringae (Ps), together with related Ps species, infects and attacks a wide range of agronomically important crops, including tomato, kiwifruit, pepper, olive and soybean, causing economic losses. Currently, chemicals and introduced resistance genes are used to protect plants against these pathogens but have limited success and may have adverse environmental impacts. Consequently, there is a pressing need to develop alternative strategies to combat bacterial disease in crops. One such strategy involves using narrow‐spectrum protein antibiotics (so‐called bacteriocins), which diverse bacteria use to compete against closely related species. Here, we demonstrate that one bacteriocin, putidacin L1 (PL1), can be expressed in an active form at high levels in Arabidopsis and in Nicotiana benthamiana in planta to provide effective resistance against diverse pathovars of Ps. Furthermore, we find that Ps strains that mutate to acquire tolerance to PL1 lose their O‐antigen, exhibit reduced motility and still cannot induce disease symptoms in PL1‐transgenic Arabidopsis. Our results provide proof‐of‐principle that the transgene‐mediated expression of a bacteriocin in planta can provide effective disease resistance to bacterial pathogens. Thus, the expression of bacteriocins in crops might offer an effective strategy for managing bacterial disease, in the same way that the genetic modification of crops to express insecticidal proteins has proven to be an extremely successful strategy for pest management. Crucially, nearly all genera of bacteria, including many plant pathogenic species, produce bacteriocins, providing an extensive source of these antimicrobial agents.     

抗真菌植物基因工程的策略和进展

所有高等植物都受多种真菌的侵害,水稻的240多种病害中真菌性痫害占90％。,可见真菌病害是世界范围内危害作物产蘑的主要因素之一,是长期以来作物育种学家一直在努力攻克的难题。目前国     

Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants

Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases.     

The Genetic and Molecular Basis of Plant Resistance to Pathogens

Plant pathogens have evolved numerous strategies to obtain nutritive materials from their host,and plants in turn have evolved the preformed physical and chemical barriers as well as sophisticated two-tiered immune system to combat pathogen attacks.Genetically, plant resistance to pathogens can be divided into qualitative and quantitative disease resistance,conditioned by major gene(s) and multiple genes with minor effects,respectively.Qualitative disease resistance has been mostly detected in plant defense against biotrophic pathogens,whereas quantitative disease resistance is involved in defense response to all plant pathogens,from biotrophs,hemibiotrophs to necrotrophs.Plant resistance is achieved through interception of pathogen-derived effectors and elicitation of defense response.In recent years,great progress has been made related to the molecular basis underlying host-pathogen interactions.In this review,we would like to provide an update on genetic and molecular aspects of plant resistance to pathogens.     

Sucrose-mediated priming of plant defense responses and broad-spectrum disease resistance by overexpression of the maize pathogenesis-related PRms protein in rice plants

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. The PRms gene encodes a fungal-inducible PR protein from maize. Here, we demonstrate that expression of PRms in transgenic rice confers broad-spectrum protection against pathogens, including fungal (Magnaporthe oryzae, Fusarium verticillioides, and Helminthosporium oryzae) and bacterial (Erwinia chrysanthemi) pathogens. The PRms-mediated disease resistance in rice plants is associated with an enhanced capacity to express and activate the natural plant defense mechanisms. Thus, PRms rice plants display a basal level of expression of endogenous defense genes in the absence of the pathogen. PRms plants also exhibit stronger and quicker defense responses during pathogen infection. We also have found that sucrose accumulates at higher levels in leaves of PRms plants. Sucrose responsiveness of rice defense genes correlates with the pathogen-responsive priming of their expression in PRms rice plants. Moreover, pretreatment of rice plants with sucrose enhances resistance to M. oryzae infection. Together, these results support a sucrose-mediated priming of defense responses in PRms rice plants which results in broad-spectrum disease resistance.     

Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens

Mycoparasitic fungi are proving to be rich sources of antifungal genes that can be utilized to genetically engineer important crops for resistance against fungal pathogens. We have transformed cotton and tobacco plants with a cDNA clone encoding a 42 kDa endochitinase from the mycoparasitic fungus, Trichoderma virens. Plants from 82 independently transformed callus lines of cotton were regenerated and analysed for transgene expression. Several primary transformants were identified with endochitinase activities that were significantly higher than the control values. Transgene integration and expression was confirmed by Southern and Northern blot analyses, respectively. The transgenic endochitinase activities were examined in the leaves of transgenic tobacco as well as in the leaves, roots, hypocotyls and seeds of transgenic cotton. Transgenic plants with elevated endochitinase activities also showed the expected 42 kDa endochitinase band in fluorescence, gel-based assays performed with the leaf extracts in both species. Homozygous T2 plants of the high endochitinase-expressing cotton lines were tested for disease resistance against a soil-borne pathogen, Rhizoctonia solani and a foliar pathogen, Alternaria alternata. Transgenic cotton plants showed significant resistance to both pathogens.     

Biotechnological potential of engineering pathogen effector proteins for use in plant disease management

A key component in the management of many diseases of crops is the use of plant disease resistance genes. However, the discovery and then sequence identification of these plant genes is challenging, whereas the characterization of the molecules that they recognize, the effector/avirulence products in pathogens, is often considerably more straight forward. Effectors are small proteins secreted by pathogens that can play major roles in modulating a plant's defense against attack. Effectors can be used to guide breeding of resistance genes, to trigger defense responses, and are part of integrated disease management strategies for crop protection. This review covers the role of effector-driven biotechnology in controlling plant diseases caused by fungi or oomycetes. Given that multi-billion dollar agriculture crops are based in some cases on plants recognizing just a handful of such effector proteins, there is considerable scope to use more fully effector proteins as a biotechnology resource in agriculture.     

Heterologous chitinase gene expression to improve plant defense against phytopathogenic fungi

Agricultural crops worldwide suffer from a vast array of fungal diseases which cause severe yield losses. Upon interaction with a pathogen, plants initiate a complex network of defense mechanisms, among which is a dramatic increase in chitinase activity. Chitinases are capable of hydrolyzing chitin-containing fungal cell walls and are therefore thought to play a major role in the plant’s response. One of the strategies to increase plant tolerance to fungal pathogens is the constitutive overexpression of proteins involved in plant-defense mechanisms. The level of protection observed in transgenic plants harboring heterologous chitinase genes varies, depending on the particular combination of enzyme, plant and pathogen tested. Nevertheless, most of these transgenic plants exhibit increased tolerance to fungal diseases relative to their non-transgenic counterparts. The combined expression of chitinases with other plant-defense proteins such as glucanases and ribosome-inactivating proteins further enhances the plant’s resistance to fungal attack. Received 29 January 1997/ Accepted in revised form 01 July 1997     

Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogen Botrytis cinerea

* Botrytis cinerea is a necrotrophic fungus that causes grey mould on a wide range of food plants, especially grapevine, tomato, soft fruits and vegetables. This disease brings about important economic losses in both pre- and postharvest crops. Successful protection of host plants against this pathogen is severely hampered by a lack of resistance genes in the hosts and the considerable phenotypic diversity of the fungus. * The aim of this study was to test whether B. cinerea manipulates the immunity-signalling pathways in plants to restore its disease. * We showed that B. cinerea caused disease in Nicotiana benthamiana through the activation of two plant signalling genes, EDS1 and SGT1, which have been shown to be essential for resistance against biotrophic pathogens; and more interestingly, virus-induced gene silencing of these two plant signalling components enhanced N. benthamiana resistance to B. cinerea. Finally, plants expressing the baculovirus antiapoptotic protein p35 were more resistant to this necrotrophic pathogen than wild-type plants. * This work highlights a new strategy used by B. cinerea to establish disease. This information is important for the design of strategies to improve plant pathogen resistance.     

Inducible expression of bacterio-opsin in transgenic tobacco and tomato plants

The development of new strategies to enhance resistance of plants to pathogens is instrumental in preventing agricultural losses. Lesion mimic, the spontaneous formation of lesions resembling hypersensitive response lesions in the absence of a pathogen, is a dramatic phenotype occasionally induced upon expression of certain transgenes in plants. These transgenes simulate the presence of a pathogen and, therefore, activate the plant anti-pathogen defense mechanisms and induce a state of systemic resistance. Lesion mimic genes have been successfully used to enhance the resistance of a number of different plants to pathogen attack. However, constitutive expression of these genes in plants is associated with the spontaneous formation of lesions on leaves and stems, reduced growth, and lower yield. We tested the possibility of using a wound-inducible promoter to control the expression of bacterio-opsin (bO), a transgene that confers a lesion mimic phenotype in tobacco and tomato plants when constitutively expressed. We found that plants with inducible expression of bO did not develop spontaneous lesions. Nevertheless, under controlled laboratory conditions, they were found to be resistant to infection by pathogens. The activation of defense mechanisms by the bO gene was not constitutive, and occurred in response to wounding or pathogen infection. Furthermore, wounding of transgenic tobacco plants resulted in the induction of systemic resistance to pathogen attack within 48 h. Our findings provide a promising initial assessment for the use of wound-inducible promoters as a new strategy to enhance pathogen resistance in transgenic crops by means of lesion mimic genes.     

Protein-protein interactions in pathogen recognition by plants

Protein-protein interactions have emerged as key determinants of whether plant encounters with pathogens result in disease or successful plant defense. Genetic interactions between plant resistance genes and pathogen avirulence genes enable pathogen recognition by plants and activate plant defense. These gene-for-gene interactions in some cases have been shown to involve direct interactions of the products of the genes, and have indicated plant intracellular localization for certain avirulence proteins. Incomplete specificity of some of the interactions in laboratory assays suggests that additional proteins might be required to confer specificity in the plant. In many cases, resistance and avirulence protein interactions have not been demonstrable, and in some cases, other plant components that interact with avirulence proteins have been found. Investigation to date has relied heavily on biochemical and cytological methods including in vitrobinding assays and immunoprecipitation, as well as genetic tools such as the yeast two-hybrid system. Observations so far, however, point to the likely requirement for multiple, interdependent protein associations in pathogen recognition, for which these techniques can be insufficient. This article reviews the protein-protein interactions that have been described in pathogen recognition by plants, and provides examples of how rapid future progress will hinge on the adoption of new and developing technologies.     

基因沉默技术在抗真菌病害中的应用和展望

真菌病害严重威胁作物的产量和品质,给国家和人民造成巨大的经济损失。尤其是引起维管束病害的土传真菌,化学农药的作用效果很不理想。利用抗性基因进行遗传育种是目前生物防治的重要手段之一,但对于缺乏抗性资源的物种,面对强大的土壤真菌病害,研究者也时常束手无策。近年来,利用RNA干扰技术发展而来的宿主诱导的基因沉默(Host induced gene silencing,HIGS)策略,在抗病虫害领域逐渐崭露头角,但由于真菌侵染的复杂多样性及土壤传播的特性,HIGS在土壤真菌病害中的应用充满神秘和挑战。本研究室近期揭示了棉花黄萎病(一种严重的土壤真菌病害)的"罪魁祸首"——大丽轮枝菌的侵染结构和侵染过程;并首次证明了宿主植物内源小RNA能够跨界进入病原菌细胞中降解致病基因表达的抗病作用;在此基础上,本研究室利用HIGS在棉花上获得了对黄萎病抗性较高的品系,成功地开辟了抗土壤黄萎真菌病害的新天地,研究结果显示出基因沉默技术在这一领域强大的应用潜力和前景。     

Plant Chitinases: Genetic Diversity and Physiological Roles

Chitinase proteins are widely distributed across diverse biological systems. Chitinases hydrolyze chitin, chitosan, lipochitooligosaccharides, peptidoglycan, arabinogalactan and glycoproteins containing N-acetylglucosamine. Analyses of genome-wide sequence and microarray expression profilings show that chitinase genes are represented by large families and the individual member genes are expressed in diverse conditions. Chitinase proteins are members in the group of the pathogenesis-related proteins that are strongly induced when host plant cells are challenged by pathogen stress and thus chitinases constitute an important arsenal of plants against fungal pathogens. Transgenic plants have been produced that overexpress chitinases alone or in conjunction with other defense-related proteins. The phenotype analyses of such plants have shown enhanced disease resistance in large number of cases. Apart from defense against pathogen stress, chitinases are implicated in relationships between plant cells and fungi (e.g., mycorrhizae associations) and bacteria (e.g., legume/Rhizobium associations). Chitinases are also involved in plant abiotic stress responses as noted for osmotic, salt, cold, wounding and heavy metal stresses. Chitinases play a role in developmental aspects of plants too (i.e., regulation of plant embryogenesis process). A detailed account of the genetic diversity and functional aspects of plant chitinases is presented in this review.     

Plants and pathogenic agents, a refined and dangerous relationship: the example of fungi

Plant-fungus interactions are highly diverse, either being beneficial to the host plant such as those leading to mycorhizal symbiosis, or very detrimental when leading to severe diseases. Since the beginning of agriculture, improvement of plant resistance to pathogens has remained a major challenge. Breeding for resistance, first conducted empirically in the past centuries, was then performed on a more theoretical basis after the statement of heredity laws by Mendel at the end of the XIXth century. As a result, most cultivated species contain various cultivars whose resistance or susceptibility to a given pathogen species depend on their interaction with various races of that pathogen. Such highly specific race-cultivar systems are particularly suited for understanding the molecular dialogue which underlies compatible (host susceptible/pathogen virulent) or incompatible (host resistant/pathogen avirulent) interactions. During the twentieth century, one of the major events that paved the way for future research was the statement by Flor [1946, 1947] of the gene-for-gene concept. Studying inheritance of the disease phenotype in the interaction between flax and Melampsora lini he showed that resistance in the host and avirulence in the pathogen are dictated by single dominant genes which correspond one to one, i.e. one resistance gene for one avirulence gene. The fact that incompatibility may depend on the presence of only one resistance (R) gene in the host and one avirulence (Avr) gene in the pathogen was fully confirmed about 40 years later. Molecular genetics and complementation experiments have allowed to isolate numerous R and Avr genes from various plant-pathogen systems, and to verify the gene-for-gene concept. These studies have enlightened the elicitor/receptor concept, formerly introduced to account for the specificity of the compatible and incompatible interactions. The present knowledge of R and Avr genes also allows to predict how such genes have evolved and how they could be used to improve disease resistance. At the beginning of the twenty first century, this remains a major challenge in view of the severe losses caused by pests and pathogens to most crops on the earth.     

Genomic advances will herald new insights into the Brassica: Leptosphaeria maculans pathosystem

The study of the relationship between plants and phytopathogenic fungi is one of the most rapidly moving fields in the plant sciences, the findings of which have contributed to the development of new strategies and technologies to protect crops. Plants employ sophisticated mechanisms to perceive and appropriately defend themselves against pathogens. A good example of plant and pathogen evolution is the gene-for-gene interaction between the fungal pathogen Leptosphaeria maculans, the causal agent of blackleg disease, and Brassica crops. This interaction has been studied at the genetic and physiological level due to its agro-economic importance. The newly available genome sequence for Brassica spp. and L.?maculans will provide the resources to study the co-evolution of this plant and pathogen. Particularly, an understanding of the co-evolution of genes responsible for virulence and resistance will lead to improved plant protection strategies for Brassica canola and provide a model to understand plant-pathogen interactions in other major crops. This review summarises the research-to-date in the study of the Brassica-L.?maculans gene-for-gene interaction, with a focus on the genetics of resistance in Brassica and the wealth of information to be gained from genome sequencing efforts.     

Induced resistance in rice against insects

Vaccinations are the mainstay of western preventive medicine, and they have been used to protect some crops against disease and insect pests. We consider rice as a model for protection using induced resistance since it is one of the most important staple crops and there have been significant new developments in: cross-resistance among rice insects, chemical pathways involved in induced resistance, sequencing the rice genome and expression of genes conferring resistance against rice insect pests. Insect attack has been found to cause lesions that kill planthopper eggs and early stages of gall midges. Damaged plants released volatiles that made them less likely to be chosen by planthoppers and more attractive to parasitoids. Chemical elicitors have been developed for dicotyledonous plants and these can induce resistance in rice, although rice does not fit models developed to explain signalling in dicots. For example, salicylic acid did not increase in rice after infection by pathogens and did not appear to be the mobile signal for induced resistance against pathogens although it was involved in induced responses to phloem-feeding insects. Jasmonic acid acted as a signal in some induced responses to pathogens as well as chewing insects. Many of the genes associated with induced resistance in rice have recently been mapped, and techniques are being developed to incorporate them into the genome of cultivated varieties. Attempts to control insect pests of rice will affect interactions with pathogens, predators and parasites, and other organisms in this agroecosystem.     

Biochemical and molecular characterization of non-host resistance keys in food crops

Generally, under normal conditions plants are resistant to many of the incompatible pathogens (viral, fungal and bacterial), and this is named “non-host resistance phenomenon”. To understand this phenomenon, different types of food crops (faba bean, squash, barley and wheat) were inoculated with compatible and incompatible pathogens. Strong resistance symptoms were observed in the non-host/incompatible pathogen combinations as compared with host/compatible pathogen combinations, which showed severe infection (susceptibility). Reactive oxygen species (ROS) mostly hydrogen peroxide and superoxide were significantly increased early 24 and 48 h after inoculation (hai) in the non-host plants comparing to the host. Antioxidant enzymes activity (catalase, polyphenol oxidase and peroxidase) were not increased at the same early time 24, 48 hai in the non-host resistant and host resistant plants, however, it increased later at 72 and 168 hai. Electrolyte leakage decreased significantly in non-host resistant and host resistant/pathogen combinations. Catalase and peroxidase genes were significantly expressed in non-host resistant and in host resistant plants as compared to the host susceptible one, which did not show expression using RT-PCR technique. Furthermore, Yr5, Yr18 and Yr26 resistant genes were identified positively using PCR in all treatments either host susceptible or non-host resistant plants in which prove that no clear role of these resistant genes in resistance. Early accumulation of ROS could have a dual roles, first role is preventing the growth or killing the pathogens early in the non-host, second, stimulating the gene appearance of related genes in addition the activition of antioxidant enzymes later on which thereby, neutralize the harmful effect of ROS and consequently suppressing disease symptoms. The new finding from this study supporting the plant breeders with new source of resistance to develop new resistant cultivars and/or stop the breakdown of resistance in resistant cultivars.     

Host‐induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce

Host‐induced gene silencing (HIGS) is an RNA interference‐based approach in which small interfering RNAs (siRNAs) are produced in the host plant and subsequently move into the pathogen to silence pathogen genes. As a proof‐of‐concept, we generated stable transgenic lettuce plants expressing siRNAs targeting potentially vital genes of Bremia lactucae, a biotrophic oomycete that causes downy mildew, the most important disease of lettuce worldwide. Transgenic plants, expressing inverted repeats of fragments of either the Highly Abundant Message #34 (HAM34) or Cellulose Synthase (CES1) genes of B. lactucae, specifically suppressed expression of these genes, resulting in greatly reduced growth and inhibition of sporulation of B. lactucae. This demonstrates that HIGS can provide effective control of B. lactucae in lettuce; such control does not rely on ephemeral resistance conferred by major resistance genes and therefore offers new opportunities for durable control of diverse diseases in numerous crops.