The role of reactive oxygen in the development of Ramularia leaf spot disease in barley seedlings.

Background and Aims: Ramularia collo-cygni is an ascomycete fungus that colonizes barley primarily as a benign endophyte, although this interaction can become pathogenic, causing the disease Ramularia leaf spot (RLS). Factors, particularly reactive oxygen species, that resulted in the transition of the fungus from endophyte to necrotrophic parasite and the development of disease symptoms were investigated. Methods: Disease development in artificially inoculated seedlings of barley varieties varying in partial resistance to RLS was related to exposure to abiotic stress prior to inoculation. Histochemical and molecular analysis determined the effect of R. collo-cygni colonization on accumulation of reactive oxygen species and antioxidant gene expression. Development of RLS on barley lines defective in antioxidant enzymes and with altered redox status or non-functional chloroplasts was compared with the accumulation of fungal biomass to determine how these factors affect disease symptom expression. Key Results: Exposure to abiotic stress increased symptom development in all susceptible and most partially resistant barley varieties, in association with greater hydrogen peroxide (H2O2) levels in leaves. Decreased activity of the antioxidant enzymes superoxide dismutase and catalase in transgenic and mutant plants had no effect on the disease transition, whereas manipulation of H2O2 levels during asymptomatic growth of the fungus increased disease symptoms in most susceptible varieties but not in partially resistant plants. Barley mutants that undergo rapid loss of green leaf area when infected by R. collo-cygni or albino mutants with non-functional chloroplasts showed reduced development of RLS symptoms. Conclusions: These results imply that in seedlings the pathogenic transition of the normally endophytic fungus R. collo-cygni does not result from senescence as such, but rather is promoted by factors that result in changes to host reactive oxygen species. Barley varieties vary in the extent to which these factors promote RLS disease.

A comparative analysis of nonhost resistance across the two Triticeae crop species wheat and barley.

BACKGROUND: Nonhost resistance (NHR) protects plants against a vast number of non-adapted pathogens which implicates a potential exploitation as source for novel disease resistance strategies. Aiming at a fundamental understanding of NHR a global analysis of transcriptome reprogramming in the economically important Triticeae cereals wheat and barley, comparing host and nonhost interactions in three major fungal pathosystems responsible for powdery mildew (Blumeria graminis ff. ssp.), cereal blast (Magnaporthe sp.) and leaf rust (Puccinia sp.) diseases, was performed. RESULTS: In each pathosystem a significant transcriptome reprogramming by adapted- or non-adapted pathogen isolates was observed, with considerable overlap between Blumeria, Magnaporthe and Puccinia. Small subsets of these general pathogen-regulated genes were identified as differentially regulated between host and corresponding nonhost interactions, indicating a fine-tuning of the general pathogen response during the course of co-evolution. Additionally, the host- or nonhost-related responses were rather specific for each pair of adapted and non-adapted isolates, indicating that the nonhost resistance-related responses were to a great extent pathosystem-specific. This pathosystem-specific reprogramming may reflect different resistance mechanisms operating against non-adapted pathogens with different lifestyles, or equally, different co-option of the hosts by the adapted isolates to create an optimal environment for infection. To compare the transcriptional reprogramming between wheat and barley, putative orthologues were identified. Within the wheat and barley general pathogen-regulated genes, temporal expression profiles of orthologues looked similar, indicating conserved general responses in Triticeae against fungal attack. However, the comparison of orthologues differentially expressed between host and nonhost interactions revealed fewer commonalities between wheat and barley, but rather suggested different host or nonhost responses in the two cereal species. CONCLUSIONS: Taken together, our results suggest independent co-evolutionary forces acting on host pathosystems mirrored by barley- or wheat-specific nonhost responses. As a result of evolutionary processes, at least for the pathosystems investigated, NHR appears to rely on rather specific plant responses.

The role of a cytosolic superoxide dismutase in barley-pathogen interactions.

Reactive oxygen species (ROS), including superoxide ( O2·-/ HO2·) and hydrogen peroxide (H2 O2 ), are differentially produced during resistance responses to biotrophic pathogens and during susceptible responses to necrotrophic and hemi-biotrophic pathogens. Superoxide dismutase (SOD) is responsible for the catalysis of the dismutation of O2·-/ HO2· to H2 O2 , regulating the redox status of plant cells. Increased SOD activity has been correlated previously with resistance in barley to the hemi-biotrophic pathogen Pyrenophora teres f. teres (Ptt, the causal agent of the net form of net blotch disease), but the role of individual isoforms of SOD has not been studied. A cytosolic CuZnSOD, HvCSD1, was isolated from barley and characterized as being expressed in tissue from different developmental stages. HvCSD1 was up-regulated during the interaction with Ptt and to a greater extent during the resistance response. Net blotch disease symptoms and fungal growth were not as pronounced in transgenic HvCSD1 knockdown lines in a susceptible background (cv. Golden Promise), when compared with wild-type plants, suggesting that cytosolic O2·-/ HO2· contributes to the signalling required to induce a defence response to Ptt. There was no effect of HvCSD1 knockdown on infection by the hemi-biotrophic rice blast pathogen Magnaporthe oryzae or the biotrophic powdery mildew pathogen Blumeria graminis f. sp. hordei, but HvCSD1 also played a role in the regulation of lesion development by methyl viologen. Together, these results suggest that HvCSD1 could be important in the maintenance of the cytosolic redox status and in the differential regulation of responses to pathogens with different lifestyles.

The genome of the emerging barley pathogen Ramularia collo-cygni.

BACKGROUND: Ramularia collo-cygni is a newly important, foliar fungal pathogen of barley that causes the disease Ramularia leaf spot. The fungus exhibits a prolonged endophytic growth stage before switching life habit to become an aggressive, necrotrophic pathogen that causes significant losses to green leaf area and hence grain yield and quality. RESULTS: The R. collo-cygni genome was sequenced using a combination of Illumina and Roche 454 technologies. The draft assembly of 30.3 Mb contained 11,617 predicted gene models. Our phylogenomic analysis confirmed the classification of this ascomycete fungus within the family Mycosphaerellaceae, order Capnodiales of the class Dothideomycetes. A predicted secretome comprising 1053 proteins included redox-related enzymes and carbohydrate-modifying enzymes and proteases. The relative paucity of plant cell wall degrading enzyme genes may be associated with the stealth pathogenesis characteristic of plant pathogens from the Mycosphaerellaceae. A large number of genes associated with secondary metabolite production, including homologs of toxin biosynthesis genes found in other Dothideomycete plant pathogens, were identified. CONCLUSIONS: The genome sequence of R. collo-cygni provides a framework for understanding the genetic basis of pathogenesis in this important emerging pathogen. The reduced complement of carbohydrate-degrading enzyme genes is likely to reflect a strategy to avoid detection by host defences during its prolonged asymptomatic growth. Of particular interest will be the analysis of R. collo-cygni gene expression during interactions with the host barley, to understand what triggers this fungus to switch from being a benign endophyte to an aggressive necrotroph.

Differential effects of lesion mimic mutants in barley on disease development by facultative pathogens.

Lesion mimic mutants display spontaneous necrotic spots and chlorotic leaves as a result of mis-regulated cell death programmes. Typically these mutants have increased resistance to biotrophic pathogens but their response to facultative fungi that cause necrotrophic diseases is less well studied. The effect of altered cell death regulation on the development of disease caused by Ramularia collo-cygni, Fusarium culmorum and Oculimacula yallundae was explored using a collection of barley necrotic (nec) lesion mimic mutants. nec8 mutants displayed lower levels of all three diseases compared to nec9 mutants, which had increased R. collo-cygni but decreased F. culmorum disease symptoms. nec1 mutants reduced disease development caused by both R. collo-cygni and F. culmorum. The severity of the nec1-induced lesion mimic phenotype and F. culmorum symptom development was reduced by mutation of the negative cell death regulator MLO. The significant reduction in R. collo-cygni symptoms caused by nec1 was completely abolished in the presence of the mlo-5 allele and both symptoms and fungal biomass were greater than in the wild-type. These results indicate that physiological pathways involved in regulation of cell death interact with one another in their effects on different fungal pathogens.

Ramularia collo-cygni--An Emerging Pathogen of Barley Crops.

Ramularia collo-cygni is the biotic factor responsible for the disease Ramularia leaf spot (RLS) of barley (Hordeum vulgare). Despite having been described over 100 years ago and being considered a minor disease in some countries, the fungus is attracting interest in the scientific community as a result of the increasing number of recorded economically damaging disease epidemics. New reports of disease spread and fungal identification using molecular diagnostics have helped redefine RLS as a global disease. This review describes recent developments in our understanding of the biology and epidemiology of the fungus, outlines advances made in the field of the genetics of both the fungus and host, and summarizes the control strategies currently available.

Contribution of the drought tolerance-related stress-responsive NAC1 transcription factor to resistance of barley to Ramularia leaf spot.

NAC proteins are plant transcription factors that are involved in tolerance to abiotic and biotic stresses, as well as in many developmental processes. Stress-responsive NAC1 (SNAC1) transcription factor is involved in drought tolerance in barley and rice, but has not been shown previously to have a role in disease resistance. Transgenic over-expression of HvSNAC1 in barley cv. Golden Promise reduced the severity of Ramularia leaf spot (RLS), caused by the fungus Ramularia collo-cygni, but had no effect on disease symptoms caused by Fusarium culmorum, Oculimacula yallundae (eyespot), Blumeria graminis f. sp. hordei (powdery mildew) or Magnaporthe oryzae (blast). The HvSNAC1 transcript was weakly induced in the RLS-susceptible cv. Golden Promise during the latter stages of R. collo-cygni symptom development when infected leaves were senescing. Potential mechanisms controlling HvSNAC1-mediated resistance to RLS were investigated. Gene expression analysis revealed no difference in the constitutive levels of antioxidant transcripts in either of the over-expression lines compared with cv. Golden Promise, nor was any difference in stomatal conductance or sensitivity to reactive oxygen species-induced cell death observed. Over-expression of HvSNAC1 delayed dark-induced leaf senescence. It is proposed that mechanisms controlled by HvSNAC1 that are involved in tolerance to abiotic stress and that inhibit senescence also confer resistance to R. collo-cygni and suppress RLS symptoms. This provides further evidence for an association between abiotic stress and senescence in barley and the development of RLS.

A change in temperature modulates defence to yellow (stripe) rust in wheat line UC1041 independently of resistance gene Yr36.

BACKGROUND: Rust diseases are of major importance in wheat production worldwide. With the constant evolution of new rust strains and their adaptation to higher temperatures, consistent and durable disease resistance is a key challenge. Environmental conditions affect resistance gene performance, but the basis for this is poorly understood. RESULTS: Here we show that a change in day temperature affects wheat resistance to Puccinia striiformis f. sp tritici (Pst), the causal agent of yellow (or stripe) rust. Using adult plants of near-isogenic lines UC1041 +/- Yr36, there was no significant difference between Pst percentage uredia coverage in plants grown at day temperatures of 18°C or 25°C in adult UC1041 + Yr36 plants. However, when plants were transferred to the lower day temperature at the time of Pst inoculation, infection increased up to two fold. Interestingly, this response was independent of Yr36, which has previously been reported as a temperature-responsive resistance gene as Pst development in adult UC1041 -Yr36 plants was similarly affected by the plants experiencing a temperature reduction. In addition, UC1041 -Yr36 plants grown at the lower temperature then transferred to the higher temperature were effectively resistant and a temperature change in either direction was shown to affect Pst development up to 8 days prior to inoculation. Results for seedlings were similar, but more variable compared to adult plants. Enhanced resistance to Pst was observed in seedlings of UC1041 and the cultivar Shamrock when transferred to the higher temperature. Resistance was not affected in seedlings of cultivar Solstice by a temperature change in either direction. CONCLUSIONS: Yr36 is effective at 18°C, refining the lower range of temperature at which resistance against Pst is conferred compared to previous studies. Results reveal previously uncharacterised defence temperature sensitivity in the UC1041 background which is caused by a change in temperature and independently of Yr36. This novel phenotype is present in some cultivars but absent in others, suggesting that Pst defence may be more stable in some cultivars than others when plants are exposed to varying temperatures.

A trade off between mlo resistance to powdery mildew and increased susceptibility of barley to a newly important disease, Ramularia leaf spot.

Ramularia leaf spot (RLS), caused by the fungus Ramularia collo-cygni, is a serious, recently emerged disease of barley in Europe and other temperate regions. This study investigated the trade off between strong resistance to powdery mildew conferred by mlo mutant alleles and increased susceptibility to RLS. In field trials and seedling tests, the presence of mlo alleles increased severity of RLS. Genetic analysis of a doubled-haploid population identified one quantitative trait locus for susceptibility to RLS, colocalizing with the mlo-11 allele for mildew resistance. The effect of mlo-11 on RLS severity was environmentally sensitive. Analysis of near-isogenic lines of different mlo mutations in various genetic backgrounds confirmed that mlo alleles increased RLS severity in seedlings and adult plants. For mlo resistance to mildew to be fully effective, the genes ROR1 and ROR2 are required. RLS symptoms were significantly reduced on mlo-5 ror double mutants but fungal DNA levels remained as high as in mlo-5 single mutants, implying that ror alleles modify the transition of the fungus from endophytism to necrotrophy. These results indicate that the widespread use of mlo resistance to control mildew may have inadvertently stimulated the emergence of RLS as a major disease of barley.

TaWIR1 contributes to post-penetration resistance to Magnaporthe oryzae, but not Blumeria graminis f. sp. tritici, in wheat.

Members of the Wheat-Induced Resistance 1 (TaWIR1) gene family are highly induced in response to a wide range of pathogens. Homologues have been identified in barley, but not in Brachypodium, whereas, in rice, only distant WIR1 candidates are known. Phylogenetic analysis placed TaWIR1a and TaWIR1b within a distinct clade of wheat transcripts, whereas TaWIR1c clustered with HvWIR1 genes. Transcripts of all three TaWIR1 genes were strongly induced by a wheat-adapted isolate of Magnaporthe oryzae. Virus-induced gene silencing of the TaWIR1 gene family had no effect on the initial penetration of epidermal cells by M. oryzae. However, following the establishment of an infection site, the fungus was able to grow more extensively within the leaf tissue, relative to control leaves, indicating a role for the TaWIR1 gene family in the cell-to-cell movement of M. oryzae. In contrast, the silencing of TaWIR1 transcripts had no effect on epidermal cell penetration by a wheat-adapted isolate of Blumeria graminis, or on the subsequent growth of hyphae. Differential transcription of TaWIR1 genes was also seen in epidermal peels, relative to the remaining leaf tissue, following inoculation with M. oryzae.

The Barley stripe mosaic virus system used for virus-induced gene silencing in cereals differentially affects susceptibility to fungal pathogens in wheat.

Barley stripe mosaic virus (BSMV) has emerged as a vector for virus-induced gene silencing (VIGS) in cereals, having been used to study a number of genes involved in resistance in both wheat and barley. However, the effects of the BSMV vector on plant physiology and disease resistance in plants remains unexplored. The BSMV inoculation control vector, BSMV:GFP was shown to cause severe viral symptoms in wheat, displaying chlorosis, leaf curling and growth inhibition typical of the symptoms seen in BSMV-infected barley. These viral symptoms were accompanied by induction of genes implicated in defense against pathogens, namely PR1, PR4, PR5, PR10 and PAL. Subsequent inoculation of BSMV:GFP-infected wheat with a wheat pathotype of Magnaporthe oryzae, the blast pathogen, resulted in decreased susceptibility. Penetration of epidermal cells and subsequent multiple cell colonization by M. oryzae was significantly reduced. This increased restriction of pathogen growth observed for BSMV:GFP infections with and without the viral coat protein gene. However, prior infection with BSMV:GFP had no effect on the development of a compatible isolate of Blumeria graminis f. sp. tritici, the causal agent of powdery mildew.

Cellular and transcriptional responses of wheat during compatible and incompatible race-specific interactions with Puccinia striiformis f. sp. tritici.

The initial stages of Puccinia striiformis f. sp. tritici (the causal agent of yellow rust in wheat) infection triggered a hypersensitive cell death (HCD) response in both compatible and Yr1-mediated incompatible interactions, although the response was earlier and more extensive in the incompatible interaction. Later stages of fungal development were only associated with an HCD response in the incompatible interaction, the HCD response being effectively suppressed in the compatible interaction. Cell autofluorescence was seen in mesophyll cells in direct contact with fungal infection hyphae (primary HCD) and in adjacent mesophyll cells (secondary HCD), indicating the activation of cell-to-cell signalling. Microarray analysis identified a number of defence-related transcripts implicated in Yr1-mediated resistance, including classical pathogenesis-related (PR) transcripts and genes involved in plant cell defence responses, such as the oxidative burst and cell wall fortification. A quantitative reverse transcriptase-polymerase chain reaction time course analysis identified a number of defence-related genes, including PR2, PR4, PR9, PR10 and WIR1 transcripts, associated with the latter stages of Yr1-mediated resistance. A meta-analysis comparison of the Yr1-regulated transcriptome with the resistance transcriptomes of the race-specific resistance gene Yr5 and the race-nonspecific adult plant resistance gene Yr39 indicated limited transcript commonality. Common transcripts were largely confined to classic PR and defence-related genes.

Wheat blast: histopathology and transcriptome reprogramming in response to adapted and nonadapted Magnaporthe isolates.

* Blast disease (causal agent Magnaporthe oryzae) has presented as a new and serious field disease of wheat in South America. Here, we investigated the responses of wheat to both adapted and nonadapted isolates of the blast fungus Magnaporthe, examining cellular defence and transcriptional changes. * Resistance towards the nonadapted isolate was associated with the formation of appositions, here termed halos, beneath attempted Magnaporthe grisea penetration sites that wheat-adapted, M. oryzae isolates were able to breach. * Transcriptome analysis indicated extensive transcriptional reprogramming following inoculation with both wheat-adapted and nonadapted isolates of Magnaporthe. Functional annotation of many of the differentially expressed transcripts classified into the categories: cell rescue and defence, plant metabolism, cellular transport and regulation of transcription (although a significant number of transcripts remain unclassified). * Defence-related transcripts induced in common by adapted and nonadapted isolates were differentially regulated in response to M. oryzae and M. grisea isolates over time. Differential expression of genes involved in cellular transport indicated the importance of this process in plant defence. Functional characterisation of these transcripts and their role in defence may eventually lead to the identification of broad-spectrum resistance mechanisms in wheat towards Magnaporthe.

Progress towards the understanding and control of sugar beet rhizomania disease.

Rhizomania is a soil-borne disease that occurs throughout the major sugar beet growing regions of the world, causing severe yield losses in the absence of effective control measures. It is caused by Beet necrotic yellow vein virus (BNYVV), which is transmitted by the obligate root-infecting parasite Polymyxa betae. BNYVV has a multipartite RNA genome with all natural isolates containing four RNA species, although some isolates have a fifth RNA. The larger RNA1 and RNA2 contain the housekeeping genes of the virus and are always required for infection, whereas the smaller RNAs are involved in pathogenicity and vector transmission. RNA5-containing isolates are restricted to Asia and some parts of Europe, and these isolates tend to be more aggressive. With no acceptable pesticides available to restrict the vector, the control of rhizomania is now achieved almost exclusively through the use of resistant cultivars. A single dominant resistance gene, Rz1, has been used to manage the disease worldwide in recent years, although this gene confers only partial resistance. More recently, new variants of BNYVV have evolved (both with and without RNA5) that are able to cause significant yield penalties on resistant cultivars. These isolates are not yet widespread, but their appearance has resulted in accelerated searches for new sources of resistance to both the virus and the vector. Combined virus and vector resistance, achieved either by conventional or transgenic breeding, offers the sugar beet industry a new approach in its continuing struggle against rhizomania.