Hypersensitive Resistance Response Research Articles

The novel avirulence effector AlAvr1 from Ascochyta lentis mediates host cultivar specificity of ascochyta blight in lentil.

Ascochyta lentis is a fungal pathogen that causes ascochyta blight in the important grain legume species lentil, but little is known about the molecular mechanism of disease or host specificity. We employed a map‐based cloning approach using a biparental A. lentis population to clone the gene AlAvr1‐1 that encodes avirulence towards the lentil cultivar PBA Hurricane XT. The mapping population was produced by mating A. lentis isolate P94‐24, which is pathogenic on the cultivar Nipper and avirulent towards Hurricane, and the isolate AlKewell, which is pathogenic towards Hurricane but not Nipper. Using agroinfiltration, we found that AlAvr1‐1 from the isolate P94‐24 causes necrosis in Hurricane but not in Nipper. The homologous corresponding gene in AlKewell, AlAvr1‐2, encodes a protein with amino acid variation at 23 sites and four of these sites have been positively selected in the P94‐24 branch of the phylogeny. Loss of AlAvr1‐1 in a gene knockout experiment produced a P94‐24 mutant strain that is virulent on Hurricane. Deletion of AlAvr1‐2 in AlKewell led to reduced pathogenicity on Hurricane, suggesting that the gene may contribute to disease in Hurricane. Deletion of AlAvr1‐2 did not affect virulence for Nipper and AlAvr1‐2 is therefore not an avirulence gene for Nipper. We conclude that the hemibiotrophic pathogen A. lentis has an avirulence effector, AlAvr1‐1, that triggers a hypersensitive resistance response in Hurricane. This is the first avirulence gene to be characterized in a legume pathogen from the Pleosporales and may help progress research on other damaging Ascochyta pathogens.

Comparison of Mineral Oil, Insecticidal, and Biopesticide Spraying Regimes for Reducing Spread of Three Potato virus Y Strains in Potato Crops.

In-field management of Potato virus Y (PVY) faces challenges caused by the changing availability and environmental acceptability of chemical agents to control aphid vectors of the virus and by proliferation of PVY strains with different symptoms and rates of spread. From 2018 to 2020, foliar spray treatments were compared in field experiments in New Brunswick, Canada, to measure effectiveness at reducing spread of PVYO, PVYN:O, and PVYNTN strains. Mineral oil, insecticide, combined oil and insecticide spray, and a biopesticide (i.e., LifeGard WG) were compared. Insecticide-only and mineral oil-only treatments were not effective, but several combined oil and insecticide treatments and biopesticide treatments significantly reduced PVY spread. The biopesticide was proportionately more effective with recombinant PVYN:O and PVYNTN strains, possibly by exciting the plant's hypersensitive resistance response, caused naturally only in cultivar 'Goldrush' by PVYO. Pesticide residue analysis showed that mineral oil increased the retention of pyrethroid insecticide in the potato foliage longer than with insecticide applied alone, which may explain the beneficial synergistic effect of combined sprays for reducing PVY spread. Tuber yields were generally unchanged in chemical insecticide treatments but were slightly lower in biopesticide treatment. The cost per PVY treatment was competitive across all effective treatments, including biopesticide; however, there was some revenue loss from lower yield with the biopesticide. This biopesticide is certified organic, however, and thus a small premium on the price for organic production could offset this yield deficit.

Reactive Oxygen Species Contribute to Symptomless, Extreme Resistance to Potato virus X in Tobacco.

Here we show that in tobacco (Nicotiana tabacum cultivar Samsun NN Rx1) the development of Rx1 gene-mediated, symptomless, extreme resistance to Potato virus X (PVX) is preceded by an early, intensive accumulation of the reactive oxygen species (ROS) superoxide (O2·-), evident between 1 and 6 h after inoculation and associated with increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activities. This suggests a direct contribution of this ROS to virus restriction during symptomless, extreme resistance. Superoxide inhibition in PVX-inoculated leaves by infiltration of antioxidants (superoxide dismutase [SOD] and catalase [CAT]) partially suppresses extreme resistance in parallel with the appearance of localized leaf necrosis resembling a hypersensitive resistance (HR) response. F1 progeny from crosses of Rx1 and ferritin overproducer (deficient in production of the ROS OH·) tobaccos also display a suppressed extreme resistance to PVX, because significantly increased virus levels are coupled to HR, suggesting a role of the hydroxyl radical (OH·) in this symptomless antiviral defense. In addition, treatment of PVX-susceptible tobacco with a superoxide-generating agent (riboflavin/methionine) results in HR-like symptoms and reduced PVX titers. Finally, by comparing defense responses during PVX-elicited symptomless, extreme resistance and HR-type resistance elicited by Tobacco mosaic virus, we conclude that defense reactions typical of an HR (e.g., induction of cell death/ROS-regulator genes and antioxidants) are early and transient in the course of extreme resistance. Our results demonstrate the contribution of early accumulation of ROS (superoxide, OH·) in limiting PVX replication during symptomless extreme resistance and support earlier findings that virus-elicited HR represents a delayed, slower resistance response than symptomless, extreme resistance.

Natural Genetic Diversity in the Potato Resistance Gene RB Confers Suppression Avoidance from Phytophthora Effector IPI-O4.

RB is a potato gene that provides resistance to a broad spectrum of genotypes of the late blight pathogen Phytophthora infestans. RB belongs to the CC-NB-LRR (coiled-coil, nucleotide-binding, leucine-rich repeat) class of resistance (R) genes, a major component of the plant immune system. The RB protein detects the presence of class I and II IPI-O effectors from P. infestans to initiate a hypersensitive resistance response, but this activity is suppressed in the presence of the Class III effector IPI-O4. Using natural genetic variation of RB within potato wild relatives, we identified two amino acids in the CC domain that alter interactions needed for suppression of resistance by IPI-O4. We have found that separate modification of these amino acids in RB can diminish or expand the resistance capability of this protein against P. infestans in both Nicotiana benthamiana and potato. Our results demonstrate that increased knowledge of the molecular mechanisms that determine resistance activation and R protein suppression by effectors can be utilized to tailor-engineer genes with the potential to provide increased durability.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

HCPro Suppression of Callose Deposition Contributes to Strain-Specific Resistance Against Potato Virus Y.

Potato virus Y (PVY; Potyviridae) is a continuing challenge for potato production owing to the increasing popularity of strain-specific resistant cultivars. Hypersensitive resistance (HR) is one type of plant defense responses to restrict virus spread. In many potato cultivars, such as cultivar Premier Russet (PR), local necrosis at the site of infection protects against the most common PVYO strain, but the HR often fails to restrain necrotic strains, which spread systemically. Here, we established the role of callose accumulation in the strain-specific resistance responses to PVY infection. We first uncovered that PVY, independent of the strain, is naturally capable of suppressing pathogenesis-related callose formation in a susceptible host. Such activity can be dissociated from viral replication by the transient expression of the viral-encoded helper component proteinase (HCPro) protein, identifying it as the pathogen elicitor. However, unlike the necrotic strain, PVYO and its corresponding HCPro are unable to block callose accumulation in resistant PR potatoes, in which we observed an abundance of callose deposition and the inability of the virus to spread. The substitution of eight amino acid residues within the HCPro C-terminal region that differ between PVYO and PVYN strains and were previously shown to be responsible for eliciting the HR response, are sufficient to restore the ability of HCProO to suppress callose accumulation, despite the resistant host background, in line with a new viral function in pathogenicity.

Biological and molecular characterization of a tomato isolate of potato virus Y (PVY) of the PVYC lineage.

An isolate of potato virus Y (PVY), PVY-H14, was collected on the island of Oahu, Hawaii, from tomato plants exhibiting stunting and necrotic lesions on leaves. PVY-H14 triggered the hypersensitive resistance response in potato cultivars King Edward and Maris Bard, typical of a PVYC strain, and was unable to infect systemically the four tested cultivars, Desiree, Maris Bard, King Edward, and Russet Norkotah. Phylogenetic analysis of H14 and the whole genomes of 31 PVY isolates of non-recombinant strains of PVY placed PVY-H14 in the same clade with PVYC and several unclassified PVY isolates from tomato and tobacco.

Pseudomonas syringae Effector AvrPphB Suppresses AvrB-Induced Activation of RPM1 but Not AvrRpm1-Induced Activation.

The Pseudomonas syringae effector AvrB triggers a hypersensitive resistance response in Arabidopsis and soybean plants expressing the disease resistance (R) proteins RPM1 and Rpg1b, respectively. In Arabidopsis, AvrB induces RPM1-interacting protein kinase (RIPK) to phosphorylate a disease regulator known as RIN4, which subsequently activates RPM1-mediated defenses. Here, we show that AvrPphB can suppress activation of RPM1 by AvrB and this suppression is correlated with the cleavage of RIPK by AvrPphB. Significantly, AvrPphB does not suppress activation of RPM1 by AvrRpm1, suggesting that RIPK is not required for AvrRpm1-induced modification of RIN4. This observation indicates that AvrB and AvrRpm1 recognition is mediated by different mechanisms in Arabidopsis, despite their recognition being determined by a single R protein. Moreover, AvrB recognition but not AvrRpm1 recognition is suppressed by AvrPphB in soybean, suggesting that AvrB recognition requires a similar molecular mechanism in soybean and Arabidopsis. In support of this, we found that phosphodeficient mutations in the soybean GmRIN4a and GmRIN4b proteins are sufficient to block Rpg1b-mediated hypersensitive response in transient assays in Nicotiana glutinosa. Taken together, our results indicate that AvrB and AvrPphB target a conserved defense signaling pathway in Arabidopsis and soybean that includes RIPK and RIN4.

Involvement of potato (Solanum tuberosum L.) MKK6 in response to potato virus Y.

Mitogen-activated protein kinase (MAPK) cascades have crucial roles in the regulation of plant development and in plant responses to stress. Plant recognition of pathogen-associated molecular patterns or pathogen-derived effector proteins has been shown to trigger activation of several MAPKs. This then controls defence responses, including synthesis and/or signalling of defence hormones and activation of defence related genes. The MAPK cascade genes are highly complex and interconnected, and thus the precise signalling mechanisms in specific plant–pathogen interactions are still not known. Here we investigated the MAPK signalling network involved in immune responses of potato (Solanum tuberosum L.) to Potato virus Y, an important potato pathogen worldwide. Sequence analysis was performed to identify the complete MAPK kinase (MKK) family in potato, and to identify those regulated in the hypersensitive resistance response to Potato virus Y infection. Arabidopsis has 10 MKK family members, of which we identified five in potato and tomato (Solanum lycopersicum L.), and eight in Nicotiana benthamiana. Among these, StMKK6 is the most strongly regulated gene in response to Potato virus Y. The salicylic acid treatment revealed that StMKK6 is regulated by the hormone that is in agreement with the salicylic acid-regulated domains found in the StMKK6 promoter. The involvement of StMKK6 in potato defence response was confirmed by localisation studies, where StMKK6 accumulated strongly only in Potato-virus-Y-infected plants, and predominantly in the cell nucleus. Using a yeast two-hybrid method, we identified three StMKK6 targets downstream in the MAPK cascade: StMAPK4_2, StMAPK6 and StMAPK13. These data together provide further insight into the StMKK6 signalling module and its involvement in plant defence.

Molecular and biological characterization of a recombinant isolate of potato virus Y from Mexico

An isolate of potato virus Y (PVY), PVY-M3, was subjected to biological characterization on potato indicators and to whole-genome sequencing. PVY-M3 induced a local and systemic hypersensitive resistance (HR) response in potato cultivar Maris Bard expressing the Nz gene while inducing no HR in potato cultivars Desiree and King Edward, carrying Ny and Nc genes, respectively. These HR responses, combined with a lack of vein necrosis in tobacco, clearly defined PVY-M3 as an isolate of the PVY(Z) strain. Recombination analysis demonstrated that PVY-M3 had a typical European PVY(NTN) genome with three recombinant junctions, and PVY(N) and PVY(O) were identified as parents.

The P25 Pathogenicity Factor of Beet necrotic yellow vein virus Targets the Sugar Beet 26S Proteasome Involved in the Induction of a Hypersensitive Resistance Response via Interaction with an F-box Protein

P25, a Beet necrotic yellow vein virus (BNYVV) pathogenicity factor, interacts with a sugar beet protein with high homology to Arabidopsis thaliana kelch repeat containing F-box family proteins (FBK) of unknown function in yeast. FBK are members of the Skp1-Cullin-F-box (SCF) complex that mediate protein degradation. Here, we confirm this sugar beet FBK-P25 interaction in vivo and in vitro and provide evidence for in planta interaction and similar subcellular distribution in Nicotiana tabacum leaf cells. P25 even interacts with an FBK from A. thaliana, a BNYVV nonhost. FBK functional classification was possible by demonstrating the interaction with A. thaliana orthologs of Skp1-like (ASK) genes, a member of the SCF E3 ligase. By means of a yeast two-hybrid bridging assay, a direct effect of P25 on SCF-complex formation involving ASK1 protein was demonstrated. FBK transient Agrobacterium tumefaciens-mediated expression in N. benthamiana leaves induced a hypersensitive response. The full-length F-box protein consists of one F-box domain followed by two kelch repeats, which alone were unable to interact with P25 in yeast and did not lead to cell-death induction. The results support the idea that P25 is involved in virus pathogenicity in sugar beet and suggest suppression of resistance response.

The Transcriptome of Compatible and Incompatible Interactions of Potato (Solanum tuberosum) with Phytophthora infestans Revealed by DeepSAGE Analysis

Late blight, caused by the oomycete Phytophthora infestans, is the most important disease of potato (Solanum tuberosum). Understanding the molecular basis of resistance and susceptibility to late blight is therefore highly relevant for developing resistant cultivars, either by marker-assissted selection or by transgenic approaches. Specific P. infestans races having the Avr1 effector gene trigger a hypersensitive resistance response in potato plants carrying the R1 resistance gene (incompatible interaction) and cause disease in plants lacking R1 (compatible interaction). The transcriptomes of the compatible and incompatible interaction were captured by DeepSAGE analysis of 44 biological samples comprising five genotypes, differing only by the presence or absence of the R1 transgene, three infection time points and three biological replicates. 30.859 unique 21 base pair sequence tags were obtained, one third of which did not match any known potato transcript sequence. Two third of the tags were expressed at low frequency (<10 tag counts/million). 20.470 unitags matched to approximately twelve thousand potato transcribed genes. Tag frequencies were compared between compatible and incompatible interactions over the infection time course and between compatible and incompatible genotypes. Transcriptional changes were more numerous in compatible than in incompatible interactions. In contrast to incompatible interactions, transcriptional changes in the compatible interaction were observed predominantly for multigene families encoding defense response genes and genes functional in photosynthesis and CO2 fixation. Numerous transcriptional differences were also observed between near isogenic genotypes prior to infection with P. infestans. Our DeepSAGE transcriptome analysis uncovered novel candidate genes for plant host pathogen interactions, examples of which are discussed with respect to possible function.

A novel type of Potato virus Y recombinant genome, determined for the genetic strain PVYE

Two Potato virus Y (PVY) isolates collected in Brazil, PVY‐AGA and PVY‐MON, were identified as recombinants between two parent genomes, PVYNTN and PVY‐NE‐11, with a novel type of genomic pattern. The new recombinants had an ordinary PVYNTN genome structure for approximately 6·7‐kb from the 5′‐end of the genome whereas the 3′‐terminal 3·0‐kb segment had two fragments of NE‐11‐like sequence separated by another small PVYNTN‐like fragment. PVY strains are defined based on the hypersensitive resistance (HR) response in potato indicators. Both PVY‐AGA and PVY‐MON isolates did not induce the HR in potato cultivars carrying Ny, Nc, or (putative) Nz genes and thus were able to overcome all known resistance genes to PVY. Only one of the two isolates, PVY‐AGA, induced a vein necrosis reaction in tobacco. The biological responses of the potato indicators and tobacco defined PVY‐MON as an isolate of the PVYE strain. To distinguish PVY‐AGA and PVY‐MON from other PVYNTN isolates, an RT‐PCR test was developed utilizing new specific primers from the capsid protein gene area and producing a characteristic 955‐bp band. Serological profiling of these PVY isolates with three monoclonal antibodies revealed an unusual reactivity, where one of the two commercial PVYN‐specific monoclonal antibodies did not recognize PVY‐AGA. The ability of these new PVY recombinants to overcome resistance genes in potato producing mild or no symptoms, combined with the lack of serological reactivity towards at least one PVYN‐specific antibody may present a significant threat posed by these isolates to seed potato production areas.

Molecular analysis of common wheat genes encoding three types of cytosolic heat shock protein 90 (Hsp90): functional involvement of cytosolic Hsp90s in the control of wheat seedling growth and disease resistance

Heat shock protein 90 (Hsp90) molecular chaperones play important roles in plant growth and responses to environmental stimuli. However, little is known about the genes encoding Hsp90s in common wheat. Here, we report genetic and functional analysis of the genes specifying cytosolic Hsp90s in this species. Three groups of homoeologous genes (TaHsp90.1, TaHsp90.2 and TaHsp90.3), encoding three types of cytosolic Hsp90, were isolated. The loci containing TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes were assigned to groups 2, 7 and 5 chromosomes, respectively. TaHsp90.1 genes exhibited higher transcript levels in the stamen than in the leaf, root and culm. TaHsp90.2 and TaHsp90.3 genes were more ubiquitously transcribed in the vegetative and reproductive organs examined. Decreasing the expression of TaHsp90.1 genes through virus-induced gene silencing (VIGS) caused pronounced inhibition of wheat seedling growth, whereas the suppression of TaHsp90.2 or TaHsp90.3 genes via VIGS compromised the hypersensitive resistance response of the wheat variety Suwon 11 to stripe rust fungus. Our work represents the first systematic determination of wheat genes encoding cytosolic Hsp90s, and provides useful evidence for the functional involvement of cytosolic Hsp90s in the control of seedling growth and disease resistance in common wheat.

The Stealth Episome: Suppression of Gene Expression on the Excised Genomic Island PPHGI-1 from Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1), which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR) leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1), revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS) of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC) was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state.

The involvement of two epoxide hydrolase genes, NbEH1.1 and NbEH1.2, of Nicotiana benthamiana in the interaction with Colletotrichum destructivum, Colletotrichum orbiculare or Pseudomonas syringae pv. tabaci.

Epoxide hydrolase hydrates epoxides to vicinal diols in the phyto-oxylipin peroxygenase pathway resulting in the production of epoxy alcohols, dihydrodiols, triols and epoxides, including many lipid epoxides associated with resistance. Two epoxide hydrolase genes from Nicotiana benthamiana L., NbEH1.1 and NbEH1.2, were amplified from coding DNA of leaves during a susceptible response to the hemibiotrophic pathogens, Colletotrichum destructivum O'Gara, Colletotrichum orbiculare Berk. and Mont. von Arx. or Pseudomonas syringae pv. tabaci Wolf and Foster, or the hypersensitive resistance response to P. syringae pv. tabaci expressing avrPto. Increases in expression of NbEH1.1 generally occurred during the late biotrophic and necrotrophic stages in the susceptible responses and before the hypersensitive response. NbEH1.2 expression was not significantly induced by C. orbiculare but was induced by C. destructivum, P. syringae pv. tabaci and P. syringae pv. tabaci expressing avrPto, although to a lesser degree than NbEH1.1. Virus-induced gene silencing of NbEH1.1 delayed the appearance of lesions for C. destructivum, reduced populations of P. syringae pv. tabaci and increased populations of P. syringae pv. tabaci expressing avrPto. The importance of epoxide hydrolase during pathogen attack may be related to its roles in detoxification, signalling, or metabolism of antimicrobial compounds.

Wheat cells accumulate a syringyl-rich lignin during the hypersensitive resistance response

The stem rust fungus Puccinia graminis f.sp. tritici is an obligately biotrophic pathogen attacking wheat ( Triticum aestivum). In compatible host/pathogen-interactions, the fungus participates in the host’s metabolism by establishing functional haustoria in the susceptible plant cells. In highly resistant wheat cultivars, fungal attack is stopped by a hypersensitive response of penetrated host cells. This mechanism of programmed cell death of single plant cells is accompanied by the intracellular accumulation of material with UV-fluorescence typical of phenolic compounds. A similar reaction can be induced in healthy wheat leaves by the application of a rust-derived elicitor. We analysed the biochemical composition of this defense-induced phenolic material. Contents of total soluble and cell wall esterified and etherified phenolic acids were determined in rust-inoculated and elicitor-treated leaves of the fully susceptible wheat cultivar Prelude and its highly resistant, near-isogenic line Prelude-Sr5. While no resistance-related changes occured in any of these fractions, the lignin content as determined by the thioglycolic acid and the acetyl bromide methods increased after elicitor treatment. Nitrobenzene oxidation revealed that the entire increase can be explained by an increase in syringyl units only. These biochemical data were confirmed by fluorescence emission spectra analyses which indicated a defense-induced enrichment of syringyl lignin for cell wall samples both from elicitor-treated wheat leaves and single host cells undergoing a hypersensitive response upon fungal penetration.

Registration of Common Bacterial Blight Resistant Dark Red Kidney Bean Germplasm Line USDK‐CBB‐15

Dark red kidney bean (Phaseolus vulgaris) germplasm line USDK-CBB-15 was developed by the USDA-ARS in cooperation with the Idaho Agricultural Experiment Station. It is a modified BC3F1:4 bulk from the cross K97305/3/SVM-2242//I9566-21-4-2/'Montcalm'. It was released in 2005 on the basis of its resistance to common bacterial blight caused by Xanthomonas axonopodis pv. phaseoli. In a greenhouse inoculation test, USDK-CBB-15 had a mean disease score of 2 on the basis of a 1 to 9 scale, in which 1 is resistant and 9 is completely susceptible. It possess both the SAP6 and SU91 markers linked with major QTL for resistance derived from Montana No.5 (via Montcalm) and tepary bean (via XAN 159), respectively. It exhibits a Type I determinate bush growth habit typical of kidney bean. Yield was 108% of 'Red Hawk' dark red kidney at Othello, Washington in 2004. Average weight of 100 seeds was 52 g and equal to Red Hawk. USDK-CBB-15 matured in 98 days, which is 2 days later than Red Hawk. It also exhibits a hypersensitive resistance response to the NL-3 strain of Bean common mosaic necrosis virus.

Natural Variation in the Pto Pathogen Resistance Gene Within Species of Wild Tomato (Lycopersicon). I. Functional Analysis of Pto Alleles

Disease resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) in the cultivated tomato, Lycopersicon esculentum, and the closely related L. pimpinellifolium is triggered by the physical interaction between plant disease resistance protein, Pto, and the pathogen avirulence protein, AvrPto. To investigate the extent to which variation in the Pto gene is responsible for naturally occurring variation in resistance to Pst, we determined the resistance phenotype of 51 accessions from seven species of Lycopersicon to isogenic strains of Pst differing in the presence of avrPto. One-third of the plants displayed resistance specifically when the pathogen expressed AvrPto, consistent with a gene-for-gene interaction. To test whether this resistance in these species was conferred specifically by the Pto gene, alleles of Pto were amplified and sequenced from 49 individuals and a subset (16) of these alleles was tested in planta using Agrobacterium-mediated transient assays. Eleven alleles conferred a hypersensitive resistance response (HR) in the presence of AvrPto, while 5 did not. Ten amino acid substitutions associated with the absence of AvrPto recognition and HR were identified, none of which had been identified in previous structure-function studies. Additionally, 3 alleles encoding putative pseudogenes of Pto were isolated from two species of Lycopersicon. Therefore, a large proportion, but not all, of the natural variation in the reaction to strains of Pst expressing AvrPto can be attributed to sequence variation in the Pto gene.

Annual Meeting, Ottawa, Ontario, 2004

Annual Meeting, Ottawa, Ontario, 2004

Identification and characterization of a novel tobacco mosaic virus resistance N gene homologue in Nicotiana tabacum plants.

Nicotiana tabacum cv. Xanthi nn plants are susceptible to infection by most tobamoviruses (TMV). However, such plants display a partial hypersensitive resistance response (HR-like response) to TMV-Cg. The genetic mechanism of the HR-like response has yet not been determined, but it may involve a gene with a function similar to that of a resistance gene, responsible for HR in resistant plants. We have cloned a gene homologous to the resistance N gene, named NH, from Nicotiana Xanthi nn plants. The coding region of NH is 5.028 base pairs (bp) long and has 82.6% nucleotide identity with the N gene. In contrast to the N gene, the NH gene lacks intron 4 and does not have sites for alternative splicing of intron 3. Analysis of its sequence revealed that NH belongs to the TIR / NSB / LRR gene class. We were able to detect stable levels of NH-transcript in Nicotiana Xanthi nn plants from 0 to 18 h post-inoculation (hpi) with TMV-Cg. Transcript levels increased slightly at 24 hpi and dropped below basal values at 48 hpi. The NH transcript was also detected in a range of resistant Nicotiana plants (N. tabacum Xanthi NN, N. glutinosa, N. glauca and N. rustica) suggesting that NH is a homologue of the N gene, rather than an allele. We have cloned and characterised the NH gene (GenBank acc. no. bankit598573 AY535010) from nn susceptible plants and postulate that this gene might be involved in the HR-like response seen in these plants.