The Association of "Pathogenesis-related" Proteins with Viral Induced Necrosis in Nicotiana sylvestris

The association of “pathogenesis-related” (PR) proteins with protection from superinfection, systemic acquired resistance and production of localized necrotic lesions was examined with a system using tobacco mosaic virus (TMV) and Nicotiana sylvestris. Leaves of N. sylvestris with a mosaic from earlier inoculation with a systemically infecting strain of TMV (TMV-C) and control plants were challenged with a necrotizing strain of TMV (TMV-P), RNA of TMV-P and turnip mosaic virus (TuMV). TMV-P virions produced localized necrotic lesions only in the dark green areas of the mosaic of TMV-C infected plants. Both RNA of TMV-P and TuMV produced localized necrotic lesions in both light green and dark green areas of the mosaic of TMV-C infected plants. All three challenge inocula produced localized necrotic lesions in previously uninoculated plants. Six days after challenge inoculation proteins were extracted from separated dark green and light green mosaic leaf tissue, and leaf material from control plants. Proteins were separated by electrophoresis in a 5 % polyacrylamide spacer gel and 10 % polyacrylamide running gel. PR proteins were found in tissue where localized necrotic lesions were produced as a result of challenge inoculation, but not in tissue that was not superinfected. PR proteins were not found in light green or dark green mosaic leaf tissue as a result of TMV-C inoculation. No PR proteins were evident in protein extracts from light green tissue challenged with TMV-P, although PR proteins were produced in dark green tissue, where necrosis occurred, from the same leaves. Systemic acquired resistance (reduction in size of lesions formed by a challenge inoculation) to TuMV or RNA of TMV-P and PR protein concentration was measured at various times in light green areas of mosaic leaves where dark green areas of the mosaic leaves had been inoculated with TMV-P. No quantitative or temporal relationship between the onset of resistance and PR protein production was found. It is concluded that PR proteins are a result of pathogen induced necrosis and not significantly involved in the mechanism(s) of viral induced resistance.     

Induction of systemic acquired resistance in cucumber by Pseudomonas syringae pv. syringae 61 HrpZPss protein

Systemic acquired resistance (SAR) is an inducible plant defense response and is effective against a broad spectrum of pathogens. Biological induction of SAR usually follows plant cell death resulting from the plant hypersensitive response (HR) elicited by an avirulent pathogen or from disease necrosis caused by a virulent pathogen. The elicitation of the HR and disease necroses by pathogenic bacteria is controlled by hrp genes. Previously, it was shown that the Pseudomonas syringae 61 (Pss61) HrpZ_Pss protein (formally harpin_Pss) elicited the HR in plants. In this study, it is shown that HrpZ_Pss induced SAR in cucumber to diverse pathogens, including the anthracnose fungus ( Colletotrichum lagenarium ), tobacco necrosis virus and the bacterial angular leaf spot bacterium ( P. s. pv. lachrymans ). A hrpH mutant of Pss61, which is defective in the secretion of HrpZ_Pss and, possibly, other protein elicitors, failed to elicit SAR. Pathogenesis-related (PR) proteins, including peroxidase, β-glucanase and chitinases, were induced in cucumber plants inoculated with Pss61, C. lagenarium or HrpZ_Pss. The induction patterns of PR proteins by HrpZ_Pss and Pss61 were the same, but were different from that induced by C. lagenarium . Interestingly, the hrpH mutant induced two of the three identified PR proteins, despite its failure to induce SAR. These results suggest that proteinaceous elicitors, such as HrpZ_Pss, that traverse the bacterial Hrp secretion pathway are involved in the biological induction of SAR and that at least some PR proteins can be induced by bacterial factors that are not controlled by hrp genes.     

Localization of Pathogenesis-Related Proteins in the Epidermis and Intercellular Spaces of Tobacco Leaves after Their Induction by Potassium Salicylate or Tobacco Mosaic Virus Infection

The localization of pathogenesis-related (PR) proteins inducedin tobacco leaves by treatment with potassium salicylate ora hypersensitive response to tobacco mosaic virus (TMV) infectionwas studied using immunochemical methods. Total PR protein levelsincreased with time after these treatments. The proportion ofPR proteins in the intercellular spaces to the total contentin the leaf discs rapidly rose in the later stage of the treatmentto about 75% on the 9th day after salicylate treatment and tomore than 80% on the 6th to 9th day after TMV inoculation. After5 days of salicylate treatment, the amounts of PR proteins inthe peeled leaf epidermis were two fold those in the mesophylltissue. Only five percent or less of the total PR proteins inthe epidermal and mesophyll tissues of salicylate-treated leaveswere detected in the isolated epidermal and mesophyll protoplasts.The sugar content in highly purified PR la, lb and lc was lessthan one mole of monosaccharide per mole of each protein. Theseresults show that the PR proteins are non-glycoproteins secretedinto the intercellular spaces. (Received January 16, 1987; Accepted July 14, 1987)     

Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation

Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.     

Induction of bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in rice by treatment with acibenzolar-S-methyl

The role of the plant defence activator, acibenzolar‐S‐methyl (ASM), in inducing resistance in rice against bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) was studied. Application of ASM induced resistance in rice to infection by Xoo. When the pathogen was clip‐inoculated to the rice plants, it caused bacterial leaf blight symptoms in the untreated control. However, in the rice plants pretreated with ASM, infection was significantly reduced. Induced systemic resistance was found to persist for up to 3 days in the pretreated rice plants. Increased phenolic content and accumulation of pathogenesis‐related (PR) proteins, viz. chitinase, β‐1,3‐glucanase and thaumatin‐like protein (TLP; PR 5) were observed in rice plants pretreated with ASM followed by inoculation with Xoo. Immunoblot analysis using rice TLP and tobacco chitinase antiserum revealed rapid induction and over‐expression of 25 and 35 kDa TLP and chitinase, respectively, in rice in response to pretreatment with ASM followed by Xoo inoculation. Based on these experiments, it is evident that induction of disease resistance in rice was accelerated following treatment with ASM.     

Induction of tomato stress protein mRNAs by ethephon, 2,6-dichloroisonicotinic acid and salicylate

To study the possible involvement of plant hormones in the synthesis of stress proteins in tomato upon inoculation with Cladosporium fulvum, we investigated the induction of mRNAs encoding PR proteins and ethylene biosynthesis enzymes by ethephon, 2,6-dichloroisonicotinic acid (INA) and salicylic acid (SA) by northern blot analysis. Ethephon slightly induced some but not all mRNAs encoding intra- and extracellular PR proteins. INA induced all PR protein mRNAs analysed, except for intracellular chitinase and extracellular PR-4. SA induced all PR protein mRNAs analyzed, except for intracellular chitinase and osmotin. None of the inducers affected the expression of ACC synthase mRNA, whereas all three induced ethylene-forming enzyme (EFE) mRNA.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - HR hypersensitive response - INA 2,6-dichloroisonicotinic acid - PR pathogenesis-related - SA salicylic acid - SAR systemic acquired resistance     

Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species

Summary Virus-induced gene silencing (VIGS) is an extremely powerful tool for plant functional genomics. We used Tobacco rattle virus (TRV)-derived VIGS vectors expressed from binary vectors within Agrobacterium to induce RNA silencing in plants. Leaf infiltration is the most common method of agroinoculation used for VIGS but this method has limitations as it is laborious for large-scale screening and some plants are difficult to infiltrate. Here we have developed a novel and simple method of agroinoculation, called 'agrodrench', where soil adjacent to the plant root is drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors. By agrodrench we successfully silenced the expression of phytoene desaturase (PDS), a 20S proteasome subunit (PB7) or Mg-protoporphyrin chelatase (Chl H) encoding genes in Nicotiana benthamiana and in economically important crops such as tomato, pepper, tobacco, potato, and Petunia, all belonging to the Solanaceae family. An important aspect of agrodrench is that it can be used for VIGS in very young seedlings, something not possible by the leaf infiltration method, which usually requires multiple fully expanded leaves for infiltration. We also demonstrated that VIGS functioned to silence target genes in plant roots. The agrodrench method of agroinoculation was more efficient than the leaf infiltration method for VIGS in roots. Agrodrench will facilitate rapid large-scale functional analysis of cDNA libraries and can also be applied to plants that are not currently amenable to VIGS technology by conventional inoculation methods.     

Effect of geminivirus infection and Bemisia infestation on accumulation of pathogenesis-related proteins in tomato

The whitefly, Bemisia tabaci biotype B, has been shown to cause pathogenesis-related (PR) proteins to accumulate in plants as a result of direct feeding, but their specific role in plant defensive systems is unclear. Our objective was to compare accumulation of tomato PR proteins (beta-1,3-glucanase, chitinase, peroxidase, P2 and P4) in response to whitefly, with or without tomato mottle virus (ToMoV) infection. Tomato PR protein response was measured over time in plants divided into three treatments: uninfected controls (with or without whiteflies) and plants infested with viruliferous (ToMoV) whiteflies. Five- to six-leaf plants were infested with approximately 5 adult whitefly per leaf. Plants were sampled prior to whitefly infestation and at 14, 28, 42, and 56 days. By 56 days, plants infested with viruliferous whiteflies had significantly more eggs (2.5-fold) and nymphs (4.5-fold) than plants with nonviruliferous whiteflies. A significant increase in the enzymatic activity of all measured PR proteins, as compared to control plants, was only seen in viruliferous whitefly-infested plants. No significant difference was observed in enzyme activities between the uninfected control plants either with or without whiteflies. The greatest differences for all PR proteins assayed were observed 42 days after treatment initiation. Protein blot analyses showed that the differences in PR protein activities among the treatments were due to changes in specific enzyme levels within the plant and were associated with concomitant increases in levels of P2 and P4 PR proteins. Under our experimental conditions, it is clear that PR protein response is much more intense when it is attacked by whiteflies carrying ToMoV than by whitefly alone.     

Suppression of the Defence-Related Oxidative Burst in Bean Leaf Tissue and Bean Suspension Cells by the Necrotrophic Pathogen Botrytis cinerea

The interaction of two selected isolates of Botrytis cinerea with bean suspension cells and bean leaf discs was compared in relation to levels of reactive oxygen intermediates (ROI). Isolate B 1.7 was arrested by a hypersensitive‐like necrosis of bean leaf tissue. According to its inability to spread and produce conidia on the bean leaf tissue it was classified as non‐aggressive. The second isolate induced a fast expanding light brownish necrosis of the leaf tissue. It was able to produce conidia on bean leaf discs and was classified as aggressive. The generation of superoxide was followed biochemically in inoculated bean cell suspensions. Both isolates induced a similar early superoxide peak approximately 18‐h post inoculation (hpi). While the non‐aggressive isolate induced a much stronger secondary superoxide burst at 33 hpi, the level of superoxide of suspension cells inoculated with the aggressive isolate was below the control level. This is the first report on the occurrence of a biphasic oxidative burst in plant cells induced by a fungal pathogen. Such a suppression of superoxide generation was also observed in bean leaf discs inoculated with the aggressive isolate. An oxidative burst‐suppressing agent was extracted from inoculated cell culture medium and determined as 2‐methyl‐succinate (2‐MS) by GC/MS analysis. The compound was detected approximately 20 hpi in the aggressive fungus–plant interaction. 2‐MS was able to suppress the hypersensitive response‐like necrosis on leaf discs as well as the second superoxide burst in suspension cells when inoculated with the non‐aggressive isolate. The early superoxide burst at 18 hpi was not affected. The results confirm the important role of enhanced production of ROI in plant resistance reactions, also for a necrotrophlike B. cinerea.     

Sprout vacuum-infiltration: a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species

Virus-induced gene silencing (VIGS) is a robust technique for identifying the functions of plant genes. Tobacco rattle virus (TRV)-mediated VIGS has been commonly used in many plants. In order to overcome the limitations of existing agroinoculation methods, we report an easy and effective method of agroinoculation for virus-induced gene silencing-sprout vacuum-infiltration (SVI). Using sprout vacuum-infiltration, we have successfully silenced the expression of phytoene desaturase and Mg-protoporphyrin chelatase genes in four important solanaceous crops, including tomato, eggplant, pepper, and Nicotiana benthamiana. The gene-silenced phenotypes are conspicuous in 1-week-old plants. The method is simple, low cost and rapid compared to other techniques such as leaf infiltration or agrodrench. It may be more practical for studying gene function in the early stages of plant growth. An important aspect of SVI is that it will be used for high-throughput VIGS screens in the future. SVI will be an effective tool to overcome the limitations of current inoculation methods and to facilitate large-scale VIGS analysis of cDNA libraries. Key message SVI is a simple, low cost agroinoculation method for VIGS. It is practical for studying the function of genes expressed in early stages of plant growth and high-throughput VIGS screens.     

Changes in apoplast protein pattern suggest an early role of cell wall structure remodelling in flagellin-triggered basal immunity

The leaf apoplast is a dynamic compartment in contact with plant pathogenic bacteria after infection. Among the very first interaction events is the receptor-mediated perception of bacterial surface molecules such as flagellin or other conserved microbe-associated molecular patterns (MAMPs). Apoplast proteins likely play a role in basal resistance (BR) or pattern-triggered immunity (PTI). Here, a proteomic approach was carried out on water soluble — potentially the most mobile — apoplast proteins from flagellin-treated tobacco (Nicotiana tabacum) leaves. As the quickness of BR/PTI seems crucial for its efficacy, samples were taken as early as 2.5 and 7 h post inoculation. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Forty-nine different proteins from 28 protein spots changed in their density compared to the water-inoculated control. Eleven protein spots appeared de novo in response to EBR induction. There are glycohydrolases and redox-active proteins besides pathogenesis-related proteins among them, predicting plant cell wall structural modifications and more direct antimicrobial effectors as earliest changes related to BR/PTI.     

Expression of stress-response proteins upon whitefly-mediated inoculation of Tomato yellow leaf curl virus in susceptible and resistant tomato plants

To better understand the nature of resistance of tomato to the whitefly (Bemisia tabaci, B biotype)-transmitted Tomato yellow leaf curl virus (TYLCV), whiteflies and TYLCV were considered as particular cases of biotic stresses and virus resistance as a particular case of successful response to these stresses. Two inbred tomato lines issued from the same breeding program that used Solanum habrochaites as a TYLCV resistance source, one susceptible and the other resistant, were used to compare the expression of key proteins involved at different stages of the plant response with stresses: mitogen-activated protein kinases (MAPKs), cellular heat shock proteins (HSPs, proteases), and pathogenesis-related (PR) proteins. The two biotic stresses-non-viruliferous whitefly feeding and virus infection with viruliferous insects--led to a slow decline in abundance of MAPKs, HSPs, and chloroplast protease FtsH (but not chloroplast protease ClpC), and induced the activities of the PR proteins, beta-1,3-glucanase, and peroxidase. This decline was less pronounced in virus-resistant than in virus-susceptible lines. Contrary to whitefly infestation and virus infection, inoculation with the fungus Sclerotinia sclerotiorum induced a rapid accumulation of the stress proteins studied, followed by a decline; the virus-susceptible and -resistant tomato lines behaved similarly in response to the fungus.     

Absolute protein quantification by LC/MS(E) for global analysis of salicylic acid-induced plant protein secretion responses

The plant cell wall is a dynamic cellular compartment consisting of a complex matrix of components that can change dramatically in response to environmental stresses. During pathogen attack, for instance, a wide spectrum of proteins that participate in various sequential processes involved in plant defense is secreted into the cell wall. In this study, a mass spectrometry, data-independent acquisition approach known as LC/MS (E) was used to assess temporal changes in the cell wall proteome in response to different levels of an endogenous inducer of plant disease defense responses, salicylic acid (SA). LC/MS (E) was used as a label-free method that enabled simultaneous protein identification and absolute femtomole quantification of each protein secreted into the extracellular matrix. A total of 74 secreted proteins were identified, 63 of which showed increased specific secretion in response to SA. A majority of this induced secretion occurred within 2 h of treatment, indicating that many proteins are involved in the early stages of plant defenses. We also identified a number of apparently nonclassically secreted proteins, suggesting that, as in many nonplant systems, Golgi/ER-independent mechanisms exist for plant protein secretion. These results provide new insight into plant apoplastic defense mechanisms and demonstrate that LC/MS (E) is a powerful tool for obtaining both relative and absolute proteome-scale quantification that can be applied to complex, time- and dose-dependent experimental designs.     

Anticipating endoplasmic reticulum stress. A novel early response before pathogenesis-related gene induction

When it is attacked by a pathogen, a plant produces a range of defense-related proteins. Many of these are synthesized by the rough endoplasmic reticulum (RER) to be secreted from the cell or deposited in vacuoles. Genes encoding endoplasmic reticulum (ER)-resident chaperones, such as the lumenal binding protein (BiP), are also induced under these conditions. Here, we show that BiP induction occurs systemically throughout the plant. Furthermore, this induction occurs rapidly and precedes expression of genes encoding pathogenesis-related (PR) proteins. The underlying signal transduction pathway was shown to be independent of the signaling molecule salicylic acid and the unfolded protein response pathway. In addition, BiP induction was independent of PR gene induction. Overproduction of BiP alone was not sufficient to cause induction of PR gene expression; however, limiting the amount of BiP in the ER lumen via superimposed ER stress inhibited the induction of PR gene expression. We propose that the induction of BiP expression during plant-pathogen interactions is required as an early response to support PR protein synthesis on the RER and that a novel signal transduction pathway exists to trigger this rapid response.     

Secretome analysis of the phytopathogen Macrophomina phaseolina cultivated in liquid medium supplemented with and without soybean leaf infusion

Macrophomina phaseolina (Tassi) Goid. is a fungal pathogen that causes root and stem rot in several economically important crops. However, most of disease control strategies have shown limited effectiveness. Despite its impact on agriculture, molecular mechanisms involved in the interaction with host plant remains poorly understood. Nevertheless, it has been proven that fungal pathogens secrete a variety of proteins and metabolites to successfully infect their host plants. In this study, a proteomic analysis of proteins secreted by M. phaseolina in culture media supplemented with soybean leaf infusion was performed. A total of 250 proteins were identified with a predominance of hydrolytic enzymes. Plant cell wall degrading enzymes together peptidases were found, probably involved in the infection process. Predicted effector proteins were also found that could induce plant cell death or suppress plant immune response. Some of the putative effectors presented similarities to known fungal virulence factors. Expression analysis of ten selected protein-coding genes showed that these genes are induced during host tissue infection and suggested their participation in the infection process. The identification of secreted proteins of M. phaseolina could be used to improve the understanding of the biology and pathogenesis of this fungus. Although leaf infusion was able to induce changes at the proteome level, it is necessary to study the changes induced under conditions that mimic the natural infection process of the soil-borne pathogen M. phaseolina to identify virulence factors.     

An efficient <Emphasis Type="Italic">Foxtail mosaic virus</Emphasis> vector system with reduced environmental risk

Background  

Plant viral vectors offer high-yield expression of pharmaceutical and commercially important proteins with a minimum of cost and preparation time. The use of Agrobacterium tumefaciens has been introduced to deliver the viral vector as a transgene to each plant cell via a simple, nonsterile infiltration technique called "agroinoculation". With agroinoculation, a full length, systemically moving virus is no longer necessary for excellent protein yield, since the viral transgene is transcribed and replicates in every infiltrated cell. Viral genes may therefore be deleted to decrease the potential for accidental spread and persistence of the viral vector in the environment.     

Multitrophic interactions of the silverleaf whitefly,host plants,competing herbivores,and phytopathogens

Our laboratory found that silverleaf whitefly (SLW; Bemisia argentifolii Bellows & Perring) feeding alters host plant physiology and chemistry. The SLW induces a number of host plant defenses, including pathogenesis-related (PR) protein accumulation (e.g., chitinases, beta-1,3-glucanases, peroxidases, chitosanases, etc.). Induction of the PR proteins by SLW feeding occurs in various plant species and varieties. The extent and type of induction is dependent on a number of factors that include host plant growing conditions, the length of time the host plant is exposed to SLW feeding, the plant variety, and SLW population densities. The appearance of PR proteins correlates well with reduced infestations of conspecific insect herbivore competitors. Greenhouse and field experiments in which herbivore competitors (cabbage looper, Trichoplusia ni; leaf miner, Liromyza trifolii) were placed on plants previously exposed to SLW feeding demonstrated behavioral differences (oviposition, feeding preferences) and reduced survival rates and development times of these insects. The interaction was asymmetrical, i.e., SLW infestations of plants previously exposed to leaf miners had little or no effect on SLW behavior (oviposition). Induction of plant-defensive proteins by SLW feeding was both local (at the feeding site) and systemic (uninfested leaves distant to the feeding site). There are interactions between diseases such as tomato mottle virus (ToMoV; a geminivirus) and the host plant and SLW. PR proteins were induced in tomato plants infected with ToMoV much as they were via non-viruliferous SLW feeding. The presence of ToMoV in tomato plants significantly increased the number of eggs produced by SLW females. Experiments using tomato plants, powdery mildew (PM), and tobacco mosaic virus (TMV) show that whitefly infestations can affect plant pathogen relationships but the effects vary among pathogen types. Enzyme analyses prior to pathogen inoculation showed that whitefly treatment significantly increased the activities of foliar chitinase and peroxidase. Evaluation of pathogen growth 3 weeks after inoculation showed that whitefly feeding significantly reduced the incidence of PM. However, TMV levels evaluated by ELISA were not significantly affected by whitefly feeding. Six weeks after inoculation with pathogens, the chitinase and peroxidase activities were still elevated in plants initially fed on by whiteflies but continuing pathogen infection had no effect on these enzymes. The possibility that geminivirus infection and/or SLW infestations isolate the host plant for the selected reproduction of the virus and the insect is discussed. Multitrophic cascade effects may contribute to the successful eruptive appearance of SLW on various crops, ranking them as a major pest. They may explain the general observation that when SLW infest a host plant there are few if any competing insect herbivores and pathogens found in the host. However, the results indicate that certain SLW-virus relationships could be mutualistic.     

Mutation of a family 8 glycosyltransferase gene alters cell wall carbohydrate composition and causes a humidity-sensitive semi-sterile dwarf phenotype in Arabidopsis

Incompatible plant-pathogen interactions result in the rapid cell death response known as hypersensitive response (HR) and activation of host defense related genes. To understand the cellular mechanism controlling defense response better, a novel pathogenesis-related (PR) gene and putative cell wall protein gene, CaTin2, was isolated through differential screening of a hot pepper cDNA library and characterized. CaTin2 gene was locally and systemically induced in hot pepper plants upon TMV-P₀ inoculation which induces HR. However, CaTin2 gene wasn't regulated by bacterial HR-specific signal pathway. The full-length cDNA for CaTin2, which is 864 nucleotides long, contained the open reading frame of 200 amino acids including cell wall targeting sequences of 26 amino acids. CaTin2 gene has no sequence similarity with other cell wall protein genes except the signal sequence and exists as only one copy in hot pepper genome. CaTin2 gene contains repeated helix-turn-helix motif consisting of 39 amino acids. CaTin2 mRNA accumulation was induced in response to various treatments such as ethylene, SA, MeJA, ABA, methyl viologen, NaCl and wounding at early time points. Subcelluar localization of CaTin2 was confirmed in the cell wall in hot pepper leaves by making CaTin2::smGFP fusion protein. The transgenic plants overexpressing CaTin2 cDNA were resistant to TMV and CMV inoculation. From these results, CaTin2 gene may encode a virus-related new cell wall protein member.