A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice

A defensive role against insect attack has been traditionally attributed to plant protease inhibitors. Here, evidence is described of the potential of a plant protease inhibitor, the potato carboxypeptidase inhibitor (PCI), to provide resistance to fungal pathogens when expressed in rice as a heterologous protein. It is shown that rice plants constitutively expressing the pci gene exhibit resistance against the economically important pathogens Magnaporthe oryzae and Fusarium verticillioides . A M. oryzae carboxypeptidase was purified by affinity chromatography and further characterized by mass spectrometry. This fungal carboxypeptidase was found to be a novel carboxypeptidase B which was fully inhibited by PCI. Overall, the results indicate that PCI exerts its antifungal activity through the inhibition of this particular fungal carboxypeptidase B. Although pci confers protection against fungal pathogens in transgenic rice, a significant cost in insect resistance is observed. Thus, the weight gain of larvae of the specialist insect Chilo suppressalis (striped stem borer) and the polyphagous insect Spodoptera littoralis (Egyptian cotton worm) fed on pci rice is significantly larger than that of insects fed on wild-type plants. Homology-based modelling revealed structural similarities between the predicted structure of the M. oryzae carboxypeptidase B and the crystal structure of insect carboxypeptidases, indicating that PCI may function not only as an inhibitor of fungal carboxypeptidases, but also as an inhibitor of insect carboxypeptidases. The potential impact of the pci gene in terms of protection against fungal and insect diseases is discussed.     

The broad-spectrum potato cyst nematode resistance gene (Hero) from tomato is the only member of a large gene family of NBS-LRR genes with an unusual amino acid repeat in the LRR region

The Hero gene of tomato is a broad spectrum resistance gene that confers a high level of resistance to all pathotypes of the potato cyst nematodes Globodera rostochiensis and partial resistance to G. pallida. The gene was identified by map-based cloning, sequencing and complementation analysis of two susceptible tomato lines with an array of 13 overlapping cosmids spanning a total distance of 135 kb. Hero encodes a protein with a nucleotide-binding site (NBS) and a leucine-rich-repeat (LRR) domain and is a member of a gene family of 14 highly homologous genes, which are clustered within a continuous 118-kb region. The isolated Hero gene displayed resistance to various G. rostochiensis pathotypes and partial resistance to G. pallida pathotype Pa2/3 in transgenic tomato lines. None of the Hero homologues conferred resistance to G. rostochiensis pathotypes. Hero can be distinguished from its homologues by the length of a compound hexanucleotide microsatellite, which codes for a charged and repetitive amino acid domain within the LRR. We propose that the expansion of this microsatellite may be involved in the evolution of the Hero resistance gene.     

Colorado potato beetle manipulates plant defenses in local and systemic leaves

Herbivore microbial associates can affect diverse interactions between plants and insect herbivores. Some insect symbionts enable herbivores to expand host plant range or to facilitate host plant use by modifying plant physiology. However, little attention has been paid to the role of herbivore-associated microbes in manipulating plant defenses. We have recently shown that Colorado potato beetle secrete the symbiotic bacteria to suppress plant defenses. The bacteria in oral secretions from the beetle hijack defense signaling pathways of host plants and the suppression of induced plant defenses benefits the beetle’s performance. While the defense suppression by the beetle-associated bacteria has been investigated in local damaged leaves, little is known about the effects of the symbiotic bacteria on the manipulation of plant defenses in systemic undamaged leaves. Here, we demonstrate that the symbiotic bacteria suppress plant defenses in both local and systemic tissues when plants are attacked by antibiotic-untreated larvae.     

Homologues of a single resistance-gene cluster in potato confer resistance to distinct pathogens: a virus and a nematode

The isolation of the nematode-resistance gene Gpa2 in potato is described, and it is demonstrated that highly homologous resistance genes of a single resistance-gene cluster can confer resistance to distinct pathogen species. Molecular analysis of the Gpa2 locus resulted in the identification of an R-gene cluster of four highly homologous genes in a region of approximately 115 kb. At least two of these genes are active: one corresponds to the previously isolated Rx1 gene that confers resistance to potato virus X, while the other corresponds to the Gpa2 gene that confers resistance to the potato cyst nematode Globodera pallida. The proteins encoded by the Gpa2 and the Rx1 genes share an overall homology of over 88% (amino-acid identity) and belong to the leucine-zipper, nucleotide-binding site, leucine-rich repeat (LZ-NBS-LRR)-containing class of plant resistance genes. From the sequence conservation between Gpa2 and Rx1 it is clear that there is a direct evolutionary relationship between the two proteins. Sequence diversity is concentrated in the LRR region and in the C-terminus. The putative effector domains are more conserved suggesting that, at least in this case, nematode and virus resistance cascades could share common components. These findings underline the potential of protein breeding for engineering new resistance specificities against plant pathogens in vitro.     

HSR203 antisense suppression in tobacco accelerates development of hypersensitive cell death

Activation of the tobacco gene hsr203 is rapid, highly localized, specific for incompatible plant-pathogen interactions, and strongly correlated with programmed cell death occurring in response to diverse pathogens. Functional characterization of hsr203 gene product has shown that HSR203 is a serine hydrolase that displays esterase activity. We show here that transgenic tobacco plants deficient in HSR203 protein exhibit an accelerated hypersensitive response when inoculated with an avirulent strain of Ralstonia solanacearum. This response was accompanied by a maximal level of cell death and a drastic inhibition of in planta bacterial growth. Transgenic plants deficient in HSR203 were also found to show increased resistance in a dosage-dependent manner to Pseudomonas syringae pv. pisi, another avirulent bacterial pathogen, and to virulent and avirulent races of Phytophthora parasitica, a fungal pathogen of tobacco, but not to different virulent bacteria. Surprisingly, expression of another hsr gene, hsr515, and that of the defence genes PR1-a and PR5, was strongly reduced in the transgenic lines. Our results suggest that hsr203 antisense suppression in tobacco can have pleiotropic effects on HR cell death and defence mechanisms, and induces increased resistance to different pathogens.     

Characterization of susceptibility and resistance responses to potato cyst nematode (Globodera spp.) infection of tomato lines in the absence and presence of the broad-spectrum nematode resistance Hero gene

The tomato Hero A gene is the only member of a multigene family that confers a high level (>80%) of resistance to all the economically important pathotypes of potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida. Although the resistance levels of transgenic tomato lines were similar to those of the tomato line LA1792 containing the introgressed Hero multigene family, transgenic potato plants expressing the tomato Hero A gene are not resistant to PCNs. Comparative microscopy studies of in vitro infected roots of PCN-susceptible tomato cv. Money Maker, the resistant breeding line LA1792, and transgenic line L10 with Ro1 pathotype have revealed no statistically significant difference in the number of juveniles invading roots. However, syncytia (specialized feeding cells) induced in LA1792 and L10 roots mostly were found to have degenerated a few days after their induction, and a few surviving syncytia were able to support only the development of males rather than females. Thus, the ratio between males and females was biased towards males on LA1792 and L10 roots. A series of changes occur in resistant plants leading to formation of a layer of necrotic cells separating the syncytium from stellar conductive tissues and this is followed by degradation of the syncytium. Although the Hero A gene is expressed in all tissues, including roots, stems, leaves, and flower buds, its expression is upregulated in roots in response to PCN infection. Moreover, the expression profiles of the Hero A correlates with the timing of death of the syncytium.     

A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant anti-effector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000.     

Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss

Plant cuticles are broadly composed of two major components: polymeric cutin and a mixture of waxes, which infiltrate the cutin matrix and also accumulate on the surface, forming an epicuticular layer. Although cuticles are thought to play a number of important physiological roles, with the most important being to restrict water loss from aerial plant organs, the relative contributions of cutin and waxes to cuticle function are still not well understood. Tomato ( Solanum lycopersicum ) fruits provide an attractive experimental system to address this question as, unlike other model plants such as Arabidopsis, they have a relatively thick astomatous cuticle, providing a poreless uniform material that is easy to isolate and handle. We identified three tomato mutants, cutin deficient 1 ( cd1 ), cd2 and cd3 , the fruit cuticles of which have a dramatic (95–98%) reduction in cutin content and substantially altered, but distinctly different, architectures. This cutin deficiency resulted in an increase in cuticle surface stiffness, and in the proportions of both hydrophilic and multiply bonded polymeric constituents. Furthermore, our data suggested that there is no correlation between the amount of cutin and the permeability of the cuticle to water, but that cutin plays an important role in protecting tissues from microbial infection. The three cd mutations were mapped to different loci, and the cloning of CD2 revealed it to encode a homeodomain protein, which we propose acts as a key regulator of cutin biosynthesis in tomato fruit.     

Genotype by environment interaction in the assessment of tolerance of partially resistant potato clones to potato cyst nematodes

Genotype by environment interactions in a number of field trials in different years are examined in relation to tolerance of potato cyst nematodes and the subsequent yield losses. A biplot technique is used to display the interaction effects graphically and facilitate identification of any patterns in the data. The results are assessed and discussed in relation to breeding strategies and the variation found between different nematode populations.     

Novel resistances to four potyviruses in tuber-bearing potato species, and temperature-sensitive expression of hypersensitive resistance to potato virus Y

Novel potyvirus resistance specificities were found in eight tested wild potato species (clones): hypersensitive resistance (HR) to potato Y potyvirus (PVY) strain groups PVY^O in Solanum megistacrolobum and S. polyadenium and PVY^N in S. stoloniferum; HR to potato V potyvirus (PW) in S. maglia, S. polyadenium, S. stoloniferum, S. sparsipilum and S. sucrense, HR to potato A potyvirus (PVA) strain group 1 in S. sucrense, and extreme resistance (ER) to PVA in S. polyadenium. S. commersonii and S. stoloniferum expressed HR to tobacco etch potyvirus (TEV) which has not been reported previously in potato species. The studied clone of S. stoloniferum expressed HR to all potyviruses and potyvirus strains tested. The clone of S. stoloniferum (2n = 48; nuclear DNA content (2C) = 3.6 pg) and S. chacoense (2n = 24; 2C=1.9 pg) were crossed and one hybrid (2n = 36; 2C = 2.9 pg) was obtained. The hybrid expressed HR to all tested potyviruses except PVA, which indicated that HR to PVA was controlled by a gene which is different from the genes (or gene) controlling HR to PVY^O, PVY^N, PVV and TEV in S. stoloniferum. On the other hand, S. chacoense and the hybrid expressed ER to cucumber mosaic cucumovirus (CMV), whereas S. stoloniferum was susceptible to CMV. All tested wild species and the six tested potato cultivars (S. tuberosum subsp. tuberosum) expressed HR to PVV. Expression of HR following infection with PVY^N induced systemic acquired resistance (SAR) in S. chacoense. HR to PVY^N in S. sparsipilum and S. sucrense and to PVY^O in potato cv. Pito was efficiently expressed at lower temperatures (16/18°C) indicated by the development of distinct necrotic lesions and/or vein necrosis in inoculated leaves, whereas the HR was rendered less effective at higher temperatures (19/24°C) which was indicated by the development of systemic infection with leaf-drop and mosaic symptoms.     

Divergent expression of cytokinin biosynthesis,signaling and catabolism genes underlying differences in feeding sites induced by cyst and root‐knot nematodes

Cyst and root‐knot nematodes are obligate parasites of economic importance with a remarkable ability to reprogram root cells into unique metabolically active feeding sites. Previous studies have suggested a role for cytokinin in feeding site formation induced by these two types of nematodes, but the mechanistic details have not yet been described. Using Arabidopsis as a host plant species, we conducted a comparative analysis of cytokinin genes in response to the beet cyst nematode (BCN), Heterodera schachtii, and the root‐knot nematode (RKN), Meloidogyne incognita. We identified distinct differences in the expression of cytokinin biosynthesis, catabolism and signaling genes in response to infection by BCN and RKN, suggesting differential manipulation of the cytokinin pathway by these two nematode species. Furthermore, we evaluated Arabidopsis histidine kinase receptor mutant lines ahk2/3, ahk2/4 and ahk3/4 in response to RKN infection. Similar to our previous studies with BCN, these lines were significantly less susceptible to RKN without compromising nematode penetration, suggesting a requirement of cytokinin signaling in RKN feeding site formation. Moreover, an analysis of ahk double mutants using CycB1;1:GUS/ahk introgressed lines revealed contrasting differences in the cytokinin receptors mediating cell cycle activation in feeding sites induced by BCN and RKN.     

The RPM1 plant disease resistance gene facilitates a rapid and sustained increase in cytosolic calcium that is necessary for the oxidative burst and hypersensitive cell death

Early events occurring during the hypersensitive resistance response (HR) were examined using the avrRpm1/RPM1 gene-for-gene interaction in Arabidopsis challenged by Pseudomonas syringae pv. tomato. Increases in cytosolic Ca2+ were measured in whole leaves using aequorin-mediated bioluminescence. During the HR a sustained increase in Ca2+ was observed which was dependent on the presence of both a functional RPM1 gene product and delivery of the cognate avirulence gene product AvrRpm1. The sequence-unrelated avirulence gene avrB, which also interacts with RPM1, generated a significantly later but similarly prolonged increase in cytosolic Ca2+. Accumulation of H2O2 at reaction sites, as revealed by electron microscopy, occurred within the same time frame as the changes in cytosolic Ca2+. The NADPH oxidase inhibitor diphenylene iodonium chloride did not affect the calcium signature, but did block H2O2 accumulation and the HR. By contrast, the calcium-channel blocker LaCl3 suppressed the increase in cytosolic Ca2+ as well as H2O2 accumulation and the HR, placing calcium elevation upstream of the oxidative burst.     

A PCR-based marker tightly linked to the nematode resistance gene,Mi, in tomato

A PCR-based codominant marker has been developed which is tightly linked to Mi, a dominant genetic locus in tomato that confers resistance to several species of root-knot nematode. DNA from tomato lines differing in nematode resistance was screened for random amplified polymorphic DNA markers linked to Mi using decamer primers. Several markers were identified. One amplified product, REX-1, obtained using a pair of decamer primers, was present as a dominant marker in all nematode-resistant tomato lines tested. REX-1 was cloned and the DNA sequences of its ends were determined and used to develop 20-mer primers. PCR amplification with the 20-mer primers produced a single amplified band in both susceptible and resistant tomato lines. The amplified bands from susceptible and resistant lines were distinguishable after cleavage with the restriction enzyme Taq I. The linkage of REX-1 to Mi was verified in an F₂ population. This marker is more tightly linked to Mi than is Aps-1, the currently-used isozyme marker, and allows screening of germplasm where the linkage between Mi and Aps-1 has been lost. Homozygous and heterozygous individuals can be distinguished and the procedure can be used for rapid, routine screening. The strategy used to obtain REX-1 is applicable to obtaining tightly-linked markers to other genetic loci. Such markers would allow rapid, concurrent screening for the segregation of several loci of interest.     

茶树CsPLK基因的鉴定与表达分析

茶树中富含茶氨酸、儿茶素和咖啡碱等重要功能成分,具有较高的价值功效,茶树在生命周期中经常遭受逆境胁迫,维生素B6(VB6)在植物体内参与逆境应答,吡哆醛激酶(pyridoxal kinase,PLK)是VB6补救途径中的关键酶。为进一步了解PLK在茶树生物合成中的功能和作用机理,该研究基于茶树基因组数据库,以龙井43为材料,采用逆转录PCR(RT-PCR)的方法从茶树中克隆出CsPLK的基因。结果表明:该基因序列长为1 179 bp,编码393个氨基酸; CsPLK蛋白和已知物种中PLK蛋白具有较高的同源性,都是核糖激酶超家族成员;通过构建pET-CsPLK载体进行原核表达,并鉴定出重组蛋白有很强的催化活性;组织表达特异性分析表明,叶中的表达量比茎、根的高,在根中最低;荧光定量PCR表示,低温诱导CsPLK上调表达,干旱诱导CsPLK下调表达,发现该基因在茶树中有明显的逆境应答,推测CsPLK在茶树的生长发育、逆境胁迫发挥重要作用。     

Histological characterization of root-knot nematode resistance in cowpea and its relation to reactive oxygen species modulation

Root-knot nematodes (Meloidogyne spp.) are sedentary endoparasiteswith a broad host range which includes economically importantcrop species. Cowpea (Vigna unguiculata L. Walp) is an importantfood and fodder legume grown in many regions where root-knotnematodes are a major problem in production fields. Severalsources of resistance to root-knot nematode have been identifiedin cowpea, including the widely used Rk gene. As part of a studyto elucidate the mechanism of Rk-mediated resistance, the histologicalresponse to avirulent M. incognita feeding of a resistant cowpeacultivar CB46 was compared with a susceptible near-isogenicline (in CB46 background). Most root-knot nematode resistancemechanisms in host plants that have been examined induced ahypersensitive response (HR). However, there was no typicalHR in resistant cowpea roots and nematodes were able to developnormal feeding sites similar to those in susceptible roots upto 9–14 d post inoculation (dpi). From 14–21 dpigiant cell deterioration was observed and the female nematodesshowed arrested development and deterioration. Nematodes failedto reach maturity and did not initiate egg laying in resistantroots. These results confirmed that the induction of resistanceis relatively late in this system. Typically in pathogen resistanceHR is closely associated with an oxidative burst (OB) in infectedtissue. The level of reactive oxygen species release in bothcompatible and incompatible reactions during early and latestages of infection was also quantified. Following a basal OBduring early infection in both susceptible and resistant roots,which was also observed in mechanically wounded root tissues,no significant OB was detected up to 14 dpi, a profile consistentwith the histological observations of a delayed resistance response.These results will be useful to design gene expression experimentsto dissect Rk-mediated resistance at the molecular level. Key words: Cowpea, histology, hypersensitive response, Meloidogyne incognita, reactive oxygen species, root-knot nematode, Vigna unguiculata Received 5 November 2007; Revised 22 January 2008 Accepted 23 January 2008     

Characterization and expression profiling of a novel cereal cyst nematode resistance gene analog in wheat

Based on the conserved regions of known resistance genes, an NBS-LRR-type CCN resistance gene analog was isolated from the CCN resistant E-10 near isogenic lines (NILs) of wheat, designated as CreZ (GenBank accession number: EU327996). It contained a complete ORF that was 2775 bp in length and encoded 924 amino acids. Sequence comparison indicated that it shared 92% nucleotide and 87% amino acid identity with those of the known CCN-resistance gene Cre3 and had similar characteristic conserved motifs to those in other established NBS-LRR disease resistance genes. The expression profiling of CreZ indicated that it was specifically expressed in the roots of resistant plants and real-time PCR analysis demonstrated that expression levels drastically increased when the plants were inoculated with cereal cyst nematodes. It could be inferred, then, that CreZ belongs to the NBS-LRR resistance gene family and is a candidate gene for potential resistance to the cereal cyst nematode. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 6, pp. 1070–1077. The text was submitted by the authors in English.