Formation Of Necrotic Lesions Research Articles

New Ophiostoma species associated with Dryocoetes himalayensis and decline of Juglans regia in Czechia

AbstractA new ophiostomatoid fungus involved in the decline of walnuts was discovered in Czechia. The fungus was isolated from necrotic wood surrounding the larval galleries of Dryocoetes himalayensis, the alien bark beetle attacking walnut trees. This insect species is rather unique within hardwood-associated bark beetle species for its aggressiveness and ability to attack apparently healthy trees. The isolated fungus, associated with an exotic insect vector is described as a new species, Ophiostoma juglandis sp. nov., based on its unique morphology and phylogenetic position. The pathogenicity tests confirmed the aggressiveness of the species to both Juglans regia and J. nigra plants, manifested by formation of extensive bleeding and necrotic lesions. The symbiotic relationship between O. juglandis and D. himalayensis is expected due to massive presence of the fungal spores on surface of adult beetles in pits forming the elytral striae. In the case of hardwood-associated ophiostomatoid fungal species, phytopathogenicity is very rarely observed. The described species is exceptional in this ability, which corresponds to the aggressive behaviour of its vector. The role of the fungus in walnut tree mortality caused by D. himalayensis is discussed.

Necrotic Liver Lesion Resolution: Another Mode of Liver Regeneration.

The liver has the great ability to regenerate after partial resection or injury, and the mechanisms underlying liver regeneration have been extensively investigated. Interestingly, acute liver injuries triggered by various etiologies are associated with the formation of necrotic lesions, and such necrotic lesions are also rapidly resolved. However, how necrotic liver lesions are repaired has not been carefully investigated until recently. In this review, we briefly summarize the spatiotemporal process of necrotic liver lesion resolution in several liver injury models including immune-mediated liver injury and drug-induced liver injury. The roles of liver nonparenchymal cells and infiltrating immune cells in controlling necrotic liver lesion resolution are discussed, which may help identify potential therapies for acute liver injury and failure.

Administration of necrostatin-1 ameliorates glucocorticoid-induced osteonecrosis of the femoral head in rats.

Glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH) is a serious complication of glucocorticoid treatment and is characterized by dysfunctional bone reconstruction at necrotic sites. Our previous study confirmed the protective potential of necrostatin-1, a selective blocker of necroptosis, in glucocorticoid-induced osteoporosis. In this study, rat models of GC-induced ONFH were established to evaluate the effects of necrostatin-1 on osteonecrotic changes and repair processes. Osteonecrosis was verified by histopathological staining. An analysis of trabecular bone architecture was performed to evaluate osteogenesis in the osteonecrotic zone. Then, necroptotic signaling molecules such as RIP1 and RIP3 were examined by immunohistochemistry. Histopathological observations indicated that necrostatin-1 administration reduced the incidence of osteonecrosis and the osteogenic response in subchondral areas. Additionally, bone histomorphometry demonstrated that necrostatin-1 intervention could restore bone reconstruction in the necrotic zone. The protective mechanism of necrostatin-1 was related to the inhibition of RIP1 and RIP3. Necrostatin-1 administration alleviated GC-induced ONFH in rats by attenuating the formation of necrotic lesions, recovering the function of osteogenesis, and suppressing glucocorticoid-induced osteocytic necroptosis by inhibiting the expression of RIP1 and RIP3.

Activation of Granulocytes in Response to a High Protein Diet Leads to the Formation of Necrotic Lesions in the Liver.

In their aspiration to become healthy, people are known to follow extreme diets. However, the acute impact on organs regulating systemic metabolism is not well characterized. Here, we investigated the acute impact of six extreme diets on the liver in mice. Most diets did not lead to clear pathology after short-term feeding. However, two weeks of feeding with a high protein diet (HPD) resulted in an acute increase of liver enzymes in the blood, indicative of liver damage. Histology revealed the formation of necrotic lesions in this organ which persisted for several weeks. Flow cytometric analysis of hepatic immune cell populations showed that HPD feeding induced activation of macrophages and neutrophils. Neutralization of the pro-inflammatory cytokine IL-1β or depletion of macrophages with clodronate-loaded liposomes or with genetic models did not ameliorate liver necrosis. In contrast, the depletion of neutrophils prevented HPD-induced hepatic inflammation. After prolonged feeding, HPD-feeding was associated with a strong increase of the cytokines IL-10 and IL-27, suggesting that anti-inflammatory mediators are activated to prevent nutrient-overload-induced damage to the liver. In summary, whereas our data indicates that most extreme diets do not have a major impact on the liver within two weeks, diets with a very high protein content may lead to severe, acute hepatic damage and should therefore be avoided.

Comparative Transcriptome and Co-Expression Network Analyses Reveal the Molecular Mechanism of Calcium-Deficiency-Triggered Tipburn in Chinese Cabbage (Brassica rapa L. ssp. Pekinensis).

Chinese cabbage tipburn is characterized by the formation of necrotic lesions on the margin of leaves, including on the insides of the leafy head. This physiological disorder is associated with a localized calcium deficiency during leaf development. However, little information is available regarding the molecular mechanisms governing Ca-deficiency-triggered tipburn. This study comprehensively analysed the transcriptomic comparison between control and calcium treatments (CK and 0 mM Ca) in Chinese cabbage to determine its molecular mechanism in tipburn. Our analysis identified that the most enriched gene ontology (GO) categories are photosynthesis, thylakoid and cofactor binding. Moreover, the KEGG pathway was most enriched in photosynthesis, carbon metabolism and carbon fixation. We also analyzed the co-expression network by functional categories and identified ten critical hub differentially expressed genes (DEGs) in each gene regulatory network (GRN). These DEGs might involve abiotic stresses, developmental processes, cell wall metabolism, calcium distribution, transcription factors, plant hormone biosynthesis and signal transduction pathways. Under calcium deficiency, CNX1, calmodulin-binding proteins and CMLs family proteins were downregulated compared to CK. In addition, plant hormones such as GA, JA, BR, Auxin and ABA biosynthesis pathways genes were downregulated under calcium treatment. Likewise, HATs, ARLs and TCP transcription factors were reported as inactive under calcium deficiency, and potentially involved in the developmental process. This work explores the specific DEGs' significantly different expression levels in 0 mM Ca and the control involved in plant hormones, cell wall developments, a light response such as chlorophylls and photosynthesis, transport metabolism and defence mechanism and redox. Our results provide critical evidence of the potential roles of the calcium signal transduction pathway and candidate genes governing Ca-deficiency-triggered tipburn in Chinese cabbage.

First report of Tomato brown rugose fruit virus in greenhouse tomato in Iran

In August 2021, symptoms of a viral-like infection were observed on the leaves and fruits of tomato plants, cvs. Emperador RZF1 (Rijk Zwaan, The Netherlands) Maxifort, Eshkol and 4129 (Seminis Vegetable Seeds, USA) in a 3.2 ha greenhouse complex in Isfahan province, Iran. Plants had been raised in Iran from imported seed. The symptoms consisted of severe mosaic and deformation of leaves (especially the upper and young leaves) and chlorotic and brown necrotic spots on the fruits (Figure 1). These symptoms resembled those of Tomato brown rugose fruit virus (ToBRFV) reported elsewhere (Salem et al., 2016). About 30% of plants were affected by symptoms across the greenhouse complex. The cultivars Eshkol and 4129 were more severely affected than others. Extracts from leaf and fruit samples induced the formation of necrotic local lesions on mechanically inoculated Nicotiana glutinosa (Figure 2) indicating the possible presence of a tobamovirus. The samples were also subjected to RT-PCR using ToBRFV-specific primers: ToBRFV-F (5'-GAAGTCCCGATGTCTGTAAGG-3') and ToBRFV-R (5'-GTGCCTACGGATGTGTATGA-3') (Ling et al., 2019). The fragment obtained from the PCR reaction (842 bp) was isolated, purified after size confirmation (by comparison with molecular weight markers), and Sanger sequenced (Sinuhe Biotech Company, Iran). Sequence comparison using BLAST verified the presence of ToBRFV in the samples. The sequence was 99.75% identical with that of ToBRFV isolate Tom1-Jo (KT383474) from Jordan. The sequence was submitted to GenBank (Accession No. OK075081). To the best of our knowledge, this is the first report of ToBRFV in Iran. Following this study, all plants were removed and destroyed from the affected greenhouse. Additional studies have been started to investigate the possible source of the outbreak. We thank the Sabzkavoshan Zistkesht Company, Iran for supporting this work.

Coagulase-negative staphylococci release a purine analog that inhibits Staphylococcus aureus virulence

Coagulase-negative staphylococci and Staphylococcus aureus colonize similar niches in mammals and conceivably compete for space and nutrients. Here, we report that a coagulase-negative staphylococcus, Staphylococcus chromogenes ATCC43764, synthesizes and secretes 6-thioguanine (6-TG), a purine analog that suppresses S. aureus growth by inhibiting de novo purine biosynthesis. We identify a 6-TG biosynthetic gene cluster in S. chromogenes and other coagulase-negative staphylococci including S. epidermidis, S. pseudintermedius and S. capitis. Recombinant S. aureus strains harbouring this operon produce 6-TG and, when used in subcutaneous co-infections in mice with virulent S. aureus USA300, protect the host from necrotic lesion formation. Used prophylactically, 6-TG reduces necrotic skin lesions in mice infected with USA300, and this effect is mediated by abrogation of toxin production. RNAseq analyses reveal that 6-TG downregulates expression of genes coding for purine biosynthesis, the accessory gene regulator (agr) and ribosomal proteins in S. aureus, providing an explanation for its effect on toxin production.

In vivo assembly of the sorgoleone biosynthetic pathway and its impact on agroinfiltrated leaves of Nicotiana benthamiana

Summary Sorgoleone, a hydrophobic compound exuded from root hair cells of Sorghum spp., accounts for much of the allelopathic activity of the genus. The enzymes involved in the biosynthesis of this compound have been identified and functionally characterized. Here, we report the successful assembly of the biosynthetic pathway and the significant impact of in vivo synthesized sorgoleone on the heterologous host Nicotiana benthamiana.A multigene DNA construct was prepared for the expression of genes required for sorgoleone biosynthesis in planta and deployed in N. benthamiana leaf tissues via Agrobacterium‐mediated transient expression. RNA‐sequencing was conducted to investigate the effects of sorgoleone, via expression of its biosynthesis pathway, on host gene expression.The production of sorgoleone in agroinfiltrated leaves as detected by gas chromatography/mass spectrometry (GC/MS) resulted in the formation of necrotic lesions, indicating that the compound caused severe phytotoxicity to these tissues. RNA‐sequencing profiling revealed significant changes in gene expression in the leaf tissues expressing the pathway during the formation of sorgoleone‐induced necrotic lesions.Transcriptome analysis suggested that the compound produced in vivo impaired the photosynthetic system as a result of downregulated gene expression for the photosynthesis apparatus and elevated expression of proteasomal genes which may play a major role in the phytotoxicity of sorgoleone.

Physiochemical and molecular features of the necrotic lesion in the Heterobasidion-Norway spruce pathosystem.

In the forest of Northern Hemisphere, the fungi Heterobasidion annosum (Fr.) Bref. s.l. causes severe root and stem rot diseases, dramatically reducing the wood quality of conifer trees. The hallmark of the host response during the infection process is the formation of necrotic lesions and reaction zones. To characterize physiochemical and molecular features of the necrotic lesion, we conducted artificial inoculations on Norway spruce plants at different developmental stages: seedlings, young and mature trees. The results were further compared against data available on the formation of reaction zones. Strong necrosis browning or enlarged necrotic lesions were observed in infected tissues. This was accompanied by elevated pH. However, the increased pH, around 6.0 in necrotic lesions, was not as high as that documented in reaction zones, above 7.0 as marked by the intensity of the blue colour in response to 2,6-dichlorophenol indophenol dye. Peroxidase activity increased in infected plants and RNA-seq analysis of necrotic lesions showed marked upregulation of defence-related genes. Our findings highlight similarities and differences between the reaction zone and necrotic lesion formation in response of conifer trees to biotic stress.

Assessment of selenium spatial distribution using μ-XFR in cowpea (Vigna unguiculata (L.) Walp.) plants: Integration of physiological and biochemical responses

Low concentrations of selenium (Se) are beneficial for plant growth. Foliar Se application at high concentrations is toxic to plants due to the formation of reactive oxygen species (ROS). This study characterized Se toxicity symptoms using X-ray fluorescence (XRF) technique in response to foliar Se application in cowpea plants. Five Se concentrations (0, 10, 25, 50, 100 e 150 g ha−1) were sprayed on leaves as sodium selenate. The visual symptoms of Se toxicity in cowpea leaves were separated into two stages: I) necrotic points with an irregular distribution and internerval chlorosis at the leaf limb border (50–100 g ha−1); II) total chlorosis with the formation of dark brown necrotic lesions (150 g ha−1). Foliar Se application at 50 g ha−1 increased photosynthetic pigments and yield. Ultrastructural analyses showed that Se foliar application above 50 g ha−1 disarranged the upper epidermis of cowpea leaves. Furthermore, Se application above 100 g ha−1 significantly increased the hydrogen peroxide concentration and lipid peroxidation inducing necrotic leaf lesions. Mapping of the elements in leaves using the XRF revealed high Se intensity, specifically in leaf necrotic lesions accompanied by calcium (Ca) as a possible attenuating mechanism of plant stress. The distribution of Se intensities in the seeds was homogeneous, without specific accumulation sites. Phosphorus (P) and sulfur (S) were found primarily located in the embryonic region. Understanding the factors involved in Se accumulation and its interaction with Ca support new preventive measurement technologies to prevent Se toxicity in plants.

Identification, Pathogenicity, and Fungicide Sensitivity of Ascochyta caulina (Teleomorph: Neocamarosporium calvescens) Associated with Black Stem on Quinoa in China.

Quinoa black stem is a new disease that affects the stems of quinoa plants and is more likely to develop under cool conditions (15 to 25°C, RH = 55 ± 2%). The typical symptoms include the formation of black necrotic lesions on the stem, which can completely wrap around the stem, causing lodging and blanking (development of 'empty' and sterile grain on the panicle). Furthermore, the pycnidia form small round protrusions on the surface of the lesions. Phylogenetic analysis revealed that representative isolates LMHS-3 and LMHS-5 were closely related to Ascochyta caulina (teleomorph: Neocamarosporium calvescens). Comprehensive morphological and molecular characterizations confirmed A. caulina as the pathogen that caused quinoa black stem. A. caulina mainly infected quinoa stems and could produce many pycnidia, but it rarely infected quinoa leaves. Pathogenicity testing showed that the most suitable temperature for the onset of quinoa black stem was from 15 to 25°C. When the temperature was increased above 30°C, the conidial germination of A. caulina became malformed, and when the temperature was decreased below 5°C, mycelium growth of A. caulina became extremely slow; thus, both extreme high and low temperatures affected the pathogenicity of A. caulina. Mancozeb and azoxystrobin fungicides were revealed to have had the strongest inhibitory effects on the conidial germination of A. caulina, and in some cases caused malformations in conidial germination. Tebuconazole and difenoconazole had the strongest inhibitory effects on A. caulina mycelial growth and less on the effects on the conidial germination. The results of the present study provide a basis for the recognition and management of quinoa black stem.

A temporal gene expression map of Chrysanthemum leaves infected with Alternaria alternata reveals different stages of defense mechanisms

Chrysanthemum (Chrysanthemum morifolium) black spot disease (CBS) poses a major threat to Chrysanthemum cultivation owing to suitable climate conditions and current lack of resistant cultivars for greenhouse cultivation. In this study, we identified a number of genes that respond to Alternaria alternata infection in resistant and susceptible Chrysanthemum cultivars. Based on RNA sequencing technology and a weighted gene coexpression network analysis (WGCNA), we constructed a model to elucidate the response of Chrysanthemum leaves to A. alternata infection at different stages and compared the mapped response of the resistant cultivar ‘Jinba’ to that of the susceptible cultivar ‘Zaoyihong’. In the early stage of infection, when lesions had not yet formed, abscisic acid (ABA), salicylic acid (SA) and EDS1-mediated resistance played important roles in the Chrysanthemum defense system. With the formation of necrotic lesions, ethylene (ET) metabolism and the Ca2+ signal transduction pathway strongly responded to A. alternata infection. During the late stage, when necrotic lesions continued to expand, members of the multidrug and toxic compound extrusion (MATE) gene family were highly expressed, and their products may be involved in defense against A. alternata invasion by exporting toxins produced by the pathogen, which plays important roles in the pathogenicity of A. alternata. Furthermore, the function of hub genes was verified by qPCR and transgenic assays. The identification of hub genes at different stages, the comparison of hub genes between the two cultivars and the highly expressed genes in the resistant cultivar ‘Jinba’ provide a theoretical basis for breeding cultivars resistant to CBS.

Evaluation of potential genetic and chemical markers for Scots pine tolerance against Heterobasidion annosum infection

Main conclusionTwo terpene compounds and four genes were identified as potential biomarkers for further evaluation for Scots pine susceptibility or tolerance against Heterobasidion annosum.Scots pine (Pinus sylvestris) is one of the main sources of timber in the boreal zone of Eurasia. Commercial pine plantations are vulnerable to root and butt rot disease caused by the fungus Heterobasidion annosum. The pathogen affects host growth rate, causes higher mortality and decreases in timber quality, resulting in considerable economic losses to forest owners. Genetic and biochemical factors contributing to Scots pine tolerance against H. annosum infection are not well understood. We assessed the predictive values of a set of potential genetic and chemical markers in a field experiment. We determined the expression levels of 25 genes and the concentrations of 36 terpenoid compounds in needles of 16 Scots pine trees randomly selected from a natural population prior to artificial infection. Stems of the same trees were artificially inoculated with H. annosum, and the length of necrotic lesions was documented 5 months post inoculation. Higher expression level of four genes included in our analysis and encoding predicted α-pinene synthase (two genes), geranyl diphosphate synthase (GPPS), and metacaspase 5 (MC5), could be associated with trees exhibiting increased levels of necrotic lesion formation in response to fungal inoculation. In contrast, concentrations of two terpenoid compounds, β-caryophyllene and α-humulene, showed significant negative correlations with the lesion size. Further studies with larger sample size will help to elucidate new biomarkers or clarify the potential of the evaluated markers for use in Scots pine disease resistance breeding programs.

The Rice SPOTTED LEAF4 (SPL4) Encodes a Plant Spastin That Inhibits ROS Accumulation in Leaf Development and Functions in Leaf Senescence.

Lesion mimic mutants (LMMs) are usually controlled by single recessive mutations that cause the formation of necrotic lesions without pathogen invasion. These genetic defects are useful to reveal the regulatory mechanisms of defense-related programmed cell death in plants. Molecular evidence has been suggested that some of LMMs are closely associated with the regulation of leaf senescence in rice (Oryza sativa). Here, we characterized the mutation underlying spotted leaf4 (spl4), which results in lesion formation and also affects leaf senescence in rice. Map-based cloning revealed that the γ ray-induced spl4-1 mutant has a single base substitution in the splicing site of the SPL4 locus, resulting in a 13-bp deletion within the encoded microtubule-interacting-and-transport (MIT) spastin protein containing an AAA-type ATPase domain. The T-DNA insertion spl4-2 mutant exhibited spontaneous lesions similar to those of the spl4-1 mutant, confirming that SPL4 is responsible for the LMM phenotype. In addition, both spl4 mutants exhibited delayed leaf yellowing during dark-induced or natural senescence. Western blot analysis of spl4 mutant leaves suggested possible roles for SPL4 in the degradation of photosynthetic proteins. Punctate signals of SPL4-fused fluorescent proteins were detected in the cytoplasm, similar to the cellular localization of animal spastin. Based on these findings, we propose that SPL4 is a plant spastin that is involved in multiple aspects of leaf development, including senescence.

Extremely strong infiltration of WT1-specific CTLs into mouse tumor by the combination vaccine with WT1-specific CTL and helper peptides.

In immunotherapy by cancer antigen-derived peptide vaccine, vaccination of cytotoxic T lymphocyte (CTL) peptide alone is common, while it remains unclear whether the addition of helper peptide vaccine to the CTL peptide vaccine is of great advantage for the enhancement of tumor immunity. In the present study, combination vaccine of Wilms’ tumor gene 1(WT1) protein-derived CTL and helper peptides induced the strong infiltration of WT1-specific CD8+ T cells into mouse tumor at frequencies of 8.8%, resulting in the formation of multiple microscopic necrotic lesions in the tumor, whereas the frequencies of WT1-specific CD8+ T cell infiltration into the tumor in the vaccination of the CTL peptide alone were only 0.32%. The majority of the infiltrated WT1-specific CD8+ T cells was effector phenotype T cells, but importantly, WT1-specific CD8+CD44+CD62L+CD103+ resident memory T cells, which could differentiate into a lot of effector phenotype T cells, existed in the tumor of mice vaccinated with the both WT1 peptides. Furthermore, T-cell receptor repertoire analysis showed the oligoclonality of these tumor infiltrating WT1 tetramer+ CD8+ T cells, and 3 clones occupied about half of them. These results indicated that WT1-specific CD4+ T cells played an essential role not only in the priming and activation of WT1-specific CD8+ T cells, but also in trafficking and infiltration of the CD8+ T cells into tumors. These results should provide us with the concept that in the clinical setting, combination vaccine of WT1-specific CTL and helper peptides would be more advantageous than the CTL peptide vaccine alone.

Propagation of cell death in dropdead1, a sorghum ortholog of the maize lls1 mutant.

We describe dropdead1-1 (ded1), an EMS-induced recessive lesion mimic mutant of sorghum. It is characterized by the formation of spreading necrotic lesions that share many attributes with those associated with the maize lethal leaf spot1 (lls1) and Arabidopsis accelerated cell death1 (acd1) mutation. We show that as in lls1, ded1 lesions are initiated by wounding and require light for continued propagation, and that loss of chloroplast integrity is responsible for ded1 cell death. Consistent with these parallels, we demonstrate that ded1 is an ortholog of lls1 and encodes pheophorbide a oxidase (PaO) with 93% identity at the protein level. The mutant ded1 allele resulted from a stop codon-inducing single base pair change in exon 6 of the sorghum ortholog of lls1. The ded1 transcript was rapidly and transiently induced after wounding and substantially elevated in leaves containing ded1 lesions. Given that PaO is a key enzyme of the chlorophyll degradation pathway, its dysfunction would result in the accumulation of pheophorbide, a potent photosensitizer that results in the production of singlet oxygen. Consistent with this, cell death associated with ded1 lesions is most likely caused by singlet oxygen as our results exclude superoxide and H2O2 from this role. We explore the signal responsible for the propagation of lesions affecting both ded1 and lls1 lesions and find that both developmental age and ethylene increase the rate of lesion expansion in both mutants.

Survey of the response of 82 domestic landraces of Zea mays to cucumber mosaic virus (CMV) reveals geographical region‐related resistance to CMV in Japan

Zea mays has been historically imported to Japan via two independent geographical routes: into southern Japan by trading with Europe in the 16th century and into northern Japan by import from North America in the 19th century. Breeding to genetically improve on quality traits and high yields has led to the current domestic landraces in each region. In a survey of 82 domestic landraces, nine out of 38 landraces originating from southern Japan showed complete immunity to cucumber mosaic virus yellow strain (CMV(Y)) without the formation of necrotic local lesions (NLLs). In contrast, three out of 44 landraces originating from northern Japan developed NLLs, but revealed no systemic spread of the virus. Due to the absence of good documentation on NLL formation in Z. mays, the response of domestic landraces Aso‐1 and Aso‐3, originating from Ibaraki in northern Japan, to a challenge with CMV(Y) and CMV(Ma‐1) was further analysed. Aso‐3 only formed NLL in response to CMV(Y) but not to CMV(Ma‐1). Moreover, in CMV(Y)‐inoculated Aso‐3, virus spread was restricted to the primary infection site and the expression of defence‐related genes was up‐regulated, whereas Aso‐1 became systemically infected with either CMV(Y) or CMV(Ma‐1). The response of Aso‐3 to CMV(Y) was inherited as a single dominant trait. Together, these results pointed towards the induction of hypersensitive response (HR)‐mediated resistance to CMV(Y) in Aso‐3. Although HR‐mediated resistance to viruses has been studied mainly in dicots, the pathosystem CMV–Z. mays may provide a model to investigate HR‐mediated resistance to viruses in monocot plants.

Cell Death Is Not Sufficient for the Restriction of Potato Virus Y Spread in Hypersensitive Response-Conferred Resistance in Potato

Hypersensitive response (HR)-conferred resistance to viral infection restricts the virus spread and is accompanied by the induction of cell death, manifested as the formation of necrotic lesions. While it is known that salicylic acid is the key component in the orchestration of the events restricting viral spread in HR, the exact function of the cell death in resistance is still unknown. We show that potato virus Y (PVY) can be detected outside the cell death zone in Ny-1-mediated HR in potato plants (cv. Rywal), observed as individual infected cells or small clusters of infected cells outside the cell death zone. By exploiting the features of temperature dependent Ny-1-mediated resistance, we confirmed that the cells at the border of the cell death zone are alive and harbor viable PVY that is able to reinitiate infection. To get additional insights into this phenomenon we further studied the dynamics of both cell death zone expansion and occurrence of viral infected cell islands outside it. We compared the response of Rywal plants to their transgenic counterparts, impaired in SA accumulation (NahG-Rywal), where the lesions occur but the spread of the virus is not restricted. We show that the virus is detected outside the cell death zone in all lesion developmental stages of HR lesions. We also measured the dynamics of lesions expansion in both genotypes. We show that while rapid lesion expansion is observed in SA-depleted plants, virus spread is even faster. On the other hand the majority of analyzed lesions slowly expand also in HR-conferred resistance opening the possibility that the infected cells are eventually engulfed by cell death zone. Taken altogether, we suggest that the HR cell death is separated from the resistance mechanisms which lead to PVY restriction in Ny-1 genetic background. We propose that HR should be regarded as a process where the dynamics of events is crucial for effectiveness of viral arrest albeit the exact mechanism conferring this resistance remains unknown.

Increased cytosine methylation at promoter of the NB-LRR class R gene RCY1 correlated with compromised resistance to cucumber mosaic virus in EMS-generated src mutants of Arabidopsis thaliana

In Arabidopsis thaliana, RESISTANCE TO CMV(Y) (RCY1), which encodes a CC-NB-LRR class protein, confers the hypersensitive response (HR) to a yellow strain of cucumber mosaic virus [CMV(Y)]. A. thaliana ecotype Col-0 overexpressing RCY1 transgenes (Col::pRCY1-HA#13) shows extreme resistance (ER) to CMV(Y). To identify novel regulatory factors involved in high RCY1 expression and ER, suppressor of RCY1-mediated resistance to CMV(Y) (src) mutants were screened from Col::pRCY1-HA#13 mutagenized with ethyl methanesulfonate. The amounts of RCY1 transcript and RCY1 protein in five src mutant lines was significantly lower than in Col::pRCY1-HA#13. In src mutants, virus systemically spread without formation of necrotic local lesions in the inoculated leaves. Genetic analysis of progenies of crosses between the src mutant and Col::pRCY1-HA#13 suggested that paramutation might have occurred in these src mutants. Indeed, cytosines in the promoters but not in other genomic regions of the RCY1 transgenes are hypermethylated in these src mutant lines. Moreover, decreased RCY1 expression was correlated with increased methylation of the RCY1 promoter region. Treatment with 5-azacytidine decreased methylation in the promoter region and partially restored RCY1 expression. Taken together, SRC may be involved in RCY1-conferred resistance to CMV(Y) through the regulation of cytosine methylation of the RCY1 promoter.

A Novel Penicillium sp. Causes Rot in Stored Sugar Beet Roots in Idaho.

Penicillium vulpinum along with a number of other fungi can lead to rot of stored sugar beet roots. However, Penicillium isolates associated with necrotic lesions on roots from a recent sugar beet storage study were determined to be different from P. vulpinum and other recognized Penicillium species. Phylogenies based on sequencing of the internal transcribed spacer (ITS)-5.8S, β-tubulin (BenA), and RNA polymerase II second largest subunit (RPB2) DNA regions indicate that these isolates are novel, but most closely related to the following Penicillium spp. in the section Fasiculata: P. aurantiogriseum, P. camemberti, and P. freii. Macro- and micromorphological data also support designating these isolates as a new species for which we propose the name, Penicillium cellarum sp. nov. Inoculation studies with the P. cellarum isolates on roots of the commercial sugar beet cultivar B-7 led to the formation of necrotic lesions 23 to 25 mm in diameter after 86 days in storage. These lesions were similar to those observed on sugar beet roots in commercial storage piles. These data indicate that P. cellarum is a pathogen which can cause root rot in stored sugar beet roots.