植物NBS类抗病基因的进化

NBS(Nucleotide-binding site)类抗病基因是植物中最重要的一类抗病基因, 其进化模式、结构特点和功能调控一直是抗病基因研究领域的热点。这类基因具有保守的结构域, 广泛存在于植物基因组中, 在不同植物基因组中数目差异较大且具有较低的表达量。此外, 同源NBS类抗病基因之间通过频繁的序列交换产生广泛的序列多样性, 且抗病基因位点具有较差的线性。依据基因之间序列交换的频率, 抗病基因可分为TypeⅠ和TypeⅡ两类。文章从抗病基因的结构、数量、分布、序列多样性、进化模式以及表达调控等方面进行了综述, 旨在为后续NBS类抗病基因的相关研究提供参考。     

Structure and evolution of plant disease resistance genes

This article reviews recent advances that shed light on plant disease resistance genes, beginning with a brief overview of their structure, followed by their genomic organization and evolution. Plant disease resistance genes have been exhaustively investigated in terms of their structural organization, sequence evolution and genome distribution. There are probably hundreds of NBS-LRR sequences and other types of R-gene-like sequences within a typical plant genome. Recent studies revealed positive selection and selective maintenance of variation in plant resistance and defence-related genes. Plant resistance genes are highly polymorphic and have diverse recognition specificities. R-genes occur as members of clustered gene families that have evolved through duplication and diversification. These genes appear to evolve more rapidly than other regions of the genome, and domains such as the leucine-rich repeat, are subject to adaptive selection     

Functional analysis of plant disease resistance genes and their downstream effectors.

Plant disease resistance (R) genes encode proteins that both determine recognition of specific pathogen-derived avirulence (Avr) proteins and initiate signal transduction pathways leading to complex defense responses. Recent developments suggest that recognition specificity of R proteins is determined by either a protein kinase domain or by a region consisting of leucine-rich repeats. R genes conferring resistance to bacterial, viral, and fungal pathogens appear to use multiple signaling pathways, some of which involve distinct proteins and others which converge upon common downstream effectors. Manipulation of R genes and their signaling pathways by transgenic expression is a promising strategy to improve disease resistance in plants.     

Structure, function and evolution of plant disease resistance genes

Gene-for-gene plant disease resistance involves two basic processes: perception of pathogen attack, followed by responses to limit disease. Perception involves receptors with high degrees of specificity for pathogen strains, which are encoded by disease resistance genes. Large repertoires of distantly related resistance (R) genes with diverse recognitional specificities are found within a single plant species. The generation of R-gene polymorphism involves gene duplication, followed by DNA-sequence divergence by point mutation, and by deletion and duplication of intragenic DNA repeats encoding blocks of leucine-rich elements. Recombination between related genes reassorts this variation to further diversify gene sequences. Pathogen pressure selects functional resistance specificities and results in the maintenance of R-gene diversity. Recent genome-sequence data reveal that the NBS-LRR (i.e. nucleotide-binding site-leucine-rich repeat) class of R genes represents as much as 1% of the Arabidopsis genome. Experimental data have shown that the LRR has a role in determination of specificity. Mutation experiments, in which R-gene signaling has been dissociated from specificity in constitutive signal mutants, have provided the potential for non-specific resistance to be expressed from pathogen-infection-induced promoters in transgenic plants.     

Development of SCAR markers to the PVY resistance gene Ryadg based on a common feature of plant disease resistance genes.

Sequence-characterized amplified regions (SCARs) were developed, based on nucleotide differences within resistance gene-like fragments isolated from a potato plant carrying the Ryadg gene, which confers extreme resistance to potato Y potyvirus (PVY). It originates from Solanum tuberosum subsp. andigena, and a susceptible potato plant. SCARs were tested using 103 potato breeding lines and cultivars with diverse genetic backgrounds derived from Europe, North America, and Japan. Two markers showed high accuracy for detection of the Ryadg gene. The SCAR marker RYSC3 was generated only in genotypes carrying Ryadg. The SCAR marker RYSC4 was detected in all genotypes carrying Ryadg but also in four PVY-susceptible genotypes. Neither marker was detected in genotypes carrying other Ry genes originating from different species than S. tuberosum subsp. andigena. Therefore, these SCAR markers should be powerful tools in marker-assisted selection for Ryadg in potato breeding programs, and should also be useful for cloning of the Ryadg gene.     

Evolution of the number of LRRs in plant disease resistance genes

The largest group of plant resistance (R) genes contain the regions that encode the nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains (NBS-LRR genes). To gain new resistance, amino acid substitutions and changes in number of the LRRs that recognize the presence of pathogens are considered important. In this study, we focus on the evolution of the number of LRRs and analyze the genome data of five plant species, Arabidopsis thaliana, Oryza sativa, Medicago truncatula, Lotus japonicus and Populus trichocarpa. We first categorized the NBS-LRR genes in each species into groups and subgroups based on the phylogenetic relationships of their NBS domain sequences. Then we estimated the evolutionary rate of the number of LRRs relative to the synonymous divergence in the NBS domain sequences by a maximum likelihood method assuming the single stepwise mutation model. The estimates ranged from 4.5 to 600 and differed between groups in the same species or between species. This indicated different roles played by different groups of the NBS-LRR genes within a species or the effects of various life history characteristics, such as generation time, of the species. We also tested the fit of the model to the data using the variance of number of LRRs in each subgroup. In some subgroups in some plants (16 out of 174 subgroups), the results of simulation using the estimated rates significantly deviated from the observed data. Those subgroups may have undergone different modes of selection from the other subgroups.     

Genomic approaches to plant disease resistance

Genomic approaches are beginning to revolutionize our understanding of plant disease resistance. Large-scale sequencing will reveal the detailed organization of resistance-gene clusters and the genetic mechanisms involved in generating new resistance specificities. Global functional analyses will elucidate the complex regulatory networks and the diversity of proteins involved in resistance and susceptibility.     

植物抗病基因克隆与功能研究进展

植物抗病基因（R基因）是分子植物病理学和植物基因工程研究的热点之一,R基因的克隆及其在抗病反应中的功能研究为揭示植物抗病机制和有效－控制植物病害奠定了基础,本文介绍了R基因的成功克隆方法和克隆新策略,对R基因编码产物的功能进行了分类分析,并对通过遗传工程途径发展R基因介导的抗病植物新品种进行了展望。     

From plant immunity to crop disease resistance

Plant diseases caused by diverse pathogens lead to a serious reduction in crop yield and threaten food security worldwide. Genetic improvement of plant immunity is considered as the most effective and sustainable approach to control crop diseases. In the last decade, our understanding of plant immunity at both molecular and genomic levels has improved greatly. Combined with advances in biotechnologies, particularly clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based genome editing, we can now rapidly identify new resistance genes and engineer disease-resistance crop plants like never before. In this review, we summarize the current knowledge of plant immunity and outline existing and new strategies for disease resistance improvement in crop plants. We also discuss existing challenges in this field and suggest directions for future studies.     

Evolving disease resistance genes

Defenses against most specialized plant pathogens are often initiated by a plant disease resistance gene. Plant genomes encode several classes of genes that can function as resistance genes. Many of the mechanisms that drive the molecular evolution of these genes are now becoming clear. The processes that contribute to the diversity of R genes include tandem and segmental gene duplications, recombination, unequal crossing-over, point mutations, and diversifying selection. Diversity within populations is maintained by balancing selection. Analyses of whole-genome sequences have and will continue to provide new insight into the dynamics of resistance gene evolution.     

The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes

Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most serious diseases of rice. Here we describe the isolation and characterization of Pib, one of the rice blast resistance genes. The Pib gene was isolated by a map-based cloning strategy. The deduced amino acid sequence of the Pib gene product contains a nucleotide binding site (NBS) and leucine-rich repeats (LRRs); thus, Pib is a member of the NBS-LRR class of plant disease resistance genes. Interestingly, a duplication of the kinase 1a, 2 and 3a motifs of the NBS region was found in the N-terminal half of the Pib protein. In addition, eight cysteine residues are clustered in the middle of the LRRs, a feature which has not been reported for other R genes. Pib gene expression was induced upon altered environmental conditions, such as altered temperatures and darkness.     

The evolution of resistance genes in multi-protein plant resistance systems

The genomic perspective aids in integrating the analysis of single resistance (R-) genes into a higher order model of complex plant resistance systems. The majority of R-genes encode a class of proteins with nucleotide binding (NB) and leucine-rich repeat (LRR) domains. Several R-proteins act in multi-protein R-complexes that mediate interaction with pathogen effectors to induce resistance signaling. The complexity of these systems seems to have resulted from multiple rounds of plant-pathogen co-evolution. R-gene evolution is thought to be facilitated by the formation of R-gene clusters, which permit sequence exchanges via recombinatorial mispairing and generate high haplotypic diversity. This pattern of evolution may also generate diversity at other loci that contribute to the R-complex. The rate of recombination at R-clusters is not necessarily homogeneous or consistent over evolutionary time: recent evidence suggests that recombination at R-clusters is increased following pathogen infection, suggesting a mechanism that induces temporary genome instability in response to extreme stress. DNA methylation and chromatin modifications may allow this instability to be conditionally regulated and targeted to specific genome regions. Knowledge of natural R-gene evolution may contribute to strategies for artificial evolution of novel resistance specificities.     

Transgenic expression of plant chitinases to enhance disease resistance

Crop plants have evolved an array of mechanisms to counter biotic and abiotic stresses. Many pathogenesis-related proteins are expressed by plants during the attack of pathogens. Advances in recombinant DNA technology and understanding of plant–microbe interactions at the molecular level have paved the way for isolation and characterization of genes encoding such proteins, including chitinases. Chitinases are included in families 18 and 19 of glycosyl hydrolases (according to www.cazy.org) and they are further categorized into seven major classes based on their aminoacid sequence homology, three-dimensional structures, and hydrolytic mechanisms of catalytic reactions. Although chitin is not a component of plant cell walls, plant chitinases are involved in development and non-specific stress responses. Also, chitinase genes sourced from plants have been successfully over-expressed in crop plants to combat fungal pathogens. Crops such as tomato, potato, maize, groundnut, mustard, finger millet, cotton, lychee, banana, grape, wheat and rice have been successfully engineered for fungal resistance either with chitinase alone or in combination with other PR proteins.     

Deployment of disease resistance genes by plant transformation - a 'mix and match' approach

Breeding for disease resistance has often resulted in the evolution of a matching virulence within the pathogen population, leading to an apparent 'breakdown' of resistance. In general, plant breeders have responded by introducing new genes for resistance, with similar consequences. This has led to 'boom-bust' cycles, where varieties possessing effective resistance are grown on an expanding acreage (boom) until matching virulence evolves and spreads within the pathogen population (bust). A variety of resistance genes have recently been identified and characterized in model systems. Together with the development of efficient plant transformation systems these genes offer an alternative means to introduce specific resistance into a crop improvement programme. However, unless the resistance genes are deployed with care, the boom-bust cycle is likely to be perpetuated.