ABSTRACT
Grapevine decline and death caused by the pathogenic fungus Fusarium oxysporum f. sp. herbemontis is among the main phytosanitary problem for viticulture in southern Brazil. The eradication of infected plants is presently the most common procedure for disease control in vineyards. Inoculation with arbuscular mycorrhizal fungi is an option to reduce or neutralize the negative impacts of soil pathogenic microorganisms, but the mechanisms of plant response involved in this process are not yet completely elucidated. In order to better understand these mechanisms, an experiment was carried out to identify proteins related to plant defence induced by the mycorrhizal fungus after infection with the pathogenic fungus. We used the grapevine rootstocks SO4 and R110 (susceptible and resistant to the pathogenic fungus, respectively) inoculated or not inoculated with the mycorrhizal fungus Rhizophagus irregularis, and inoculated or not inoculated with Fusarium oxysporum f. sp. herbemontis. Growth of the rootstocks’ shoot and root and presence of pathogenic symptoms were evaluated. The protein profiles of roots were characterized by two-dimensional electrophoresis and proteins were identified using mass spectrometry. The grapevine rootstocks inoculated with R. irregularis had higher biomass production and lower level of pathogenic symptoms. The R110 rootstock differentially accumulated 73 proteins, while SO4 accumulated 59 proteins. Nine plant-defence proteins were expressed by SO4 rootstock, and six were expressed by R110 rootstock plants. The results confirm the effect of mycorrhizal fungi in plant growth promotion and their potential for biological control against soil pathogenic fungus. Protein expression is dependent on rootstock characteristics and on the combination of plant material with the fungi.
vitis; mycorrhiza; pathogenic fungus; defence response; proteins
INTRODUCTION
The main phytosanitary problem in southern Brazil is grapevine decline and death, caused by the soil pathogenic fungus Fusarium oxysporum Schlechtendahl emend. Snyder & Hansen f. sp. herbemontis Tochetto (FOH). The occurrence of this fungus is widespread in soils from this region, and the use of resistant grapevine rootstock is the main practice for control of the pathogenic attack on plants (Garrido et al., 2004Garrido LR, Sônego OR, Gomes VN. Fungos associados com o declínio e morte de videiras no Estado do Rio Grande do Sul. Fitopatol Bras. 2004;29:322-4. doi:10.1590/S0100-41582004000300016
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; Sônego et al., 2005)Sônego OR, Garrido LR, Grigoletti Júnior A. Principais doenças fúngicas da videira no Sul do Brasil. Bento Gonçalves: Embrapa-CNPUV; 2005. (Circular técnica, 56).. Among the grapevine rootstocks available in southern Brazil, the SO4 rootstock (Vitis berlandieri × V. riparia) was commonly used in grapevine production due to its adaptability to different types of soils and climate, as well as to its vigour, which ensures high yield and fruit quality. However, SO4 was replaced, due to its susceptibility to FOH (Andrade et al., 1993Andrade ER, Schuck E, Dal Bó MA. Avaliação da Resistência de Vitis spp. a Fusarium oxysporum f. sp. herbemontis em condições controladas. Pesq Agropec Bras. 1993;28:1287-90.; Dalla Costa et al., 2010Dalla Costa M, Lovato PE, Sete PB. Micorrização e indução de quitinases e β-1,3-glucanases e resistência à fusariose em porta-enxerto de videira. Pesq Agropec Bras. 2010;45:376-83. doi:10.1590/S0100-204X2010000400005
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; Cangahuala-Inocente et al., 2011)Cangahuala-Inocente GC, Da Silva MF, Johnson JM, Manga A, van Tuinen D, Henry C, Lovato PE, Dumas-Gaudot E. Arbuscular mycorrhizal symbiosis elicits proteome responses opposite of P-starvation in SO4 grapevine rootstock upon root colonisation with two Glomus species. Mycorrhiza. 2011;21:473-93. doi:10.1007/s00572-010-0352-0
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, by Paulsen 1103, R99 and R110 (V. berlandieri × V. rupestris), and VR043-43 (V. vinifera × V. rotundifolia) grapevine rootstocks, resistant to FOH, which are currently the most recommended rootstocks in southern Brazil (Sônego et al., 2005Sônego OR, Garrido LR, Grigoletti Júnior A. Principais doenças fúngicas da videira no Sul do Brasil. Bento Gonçalves: Embrapa-CNPUV; 2005. (Circular técnica, 56).; Souza et al., 2013)Souza EL, Dalbó MA, Rosier JP, Dambrós RN, Andrade ER, Arioli CJ, Parizotto C, Brighenti E, Bruna ED. Uva. In: Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina, editor. Avaliação de cultivares para o estado de Santa Catarina 2013/2014. Florianópolis: 2013. p.60-2. (Boletim técnico, 162).. These rootstocks have low root development, and consequently low yield, but they have a long production cycle (Dalla Costa et al., 2010)Dalla Costa M, Lovato PE, Sete PB. Micorrização e indução de quitinases e β-1,3-glucanases e resistência à fusariose em porta-enxerto de videira. Pesq Agropec Bras. 2010;45:376-83. doi:10.1590/S0100-204X2010000400005
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.
A sustainable option to prevent diseases from soil pathogens is the association between plants and beneficial microorganisms, especially when combined with other types of biological control (Azcón-Aguilar and Barea, 1996Azcón-Aguilar C, Barea JM. Arbuscular mycorrhizal and biological control of soil-borne plant pathogens - an overview of the mechanisms involved. Mycorrhiza. 1996;6:457-64. doi:10.1007/s005720050147
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; Hol et al., 2014Hol WHG, de Boer W, Medina A. Beneficial interactions in the rhizosphere. In: Dighton J, Krumins JA, editors. Interactions in soil: promoting plant growth. Dordrecht: Springer; 2014. p.59-80. doi:10.1007/978-94-017-8890-8_3
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). Arbuscular mycorrhizal fungi (AMF) are soil fungi that establish a mutualistic interaction with plant roots. This symbiosis benefits plants by increasing mineral and water uptake, and also improves resistance against abiotic and biotic stress (Smith and Read, 2008Smith SE, Read DJ. Mycorrhizal symbiosis. 3rd ed. San Diego: Academic Press; 2008.). For this reason, AMF are known as a biological control tool for use in agriculture (Gianinazzi et al., 2010Gianinazzi S, Gallotte A, Binet M-N, van Tuinen D, Redecker D, Wipf D. Agroecology: the key role of arbuscular mycorrhizas in ecosystem services. Mycorrhiza. 2010;20:519-30. doi:10.1007/s00572-010-0333-3
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; Trouvelot et al., 2015Trouvelot S, Bonneau L, Redecker D, van Tuienen D, Adrian M, Wipf D. Arbuscular mycorrhiza symbiosis in viticulture: a review. Agron Sustain Dev. 2015;35:1449-67. doi:10.1007/s13593-015-0329-7
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). Many studies have shown that AMF improve plant resistance to soil pathogenic fungi, including Fusarium in grapevine rootstocks and tomato plants (Dalla Costa et al., 2010Dalla Costa M, Lovato PE, Sete PB. Micorrização e indução de quitinases e β-1,3-glucanases e resistência à fusariose em porta-enxerto de videira. Pesq Agropec Bras. 2010;45:376-83. doi:10.1590/S0100-204X2010000400005
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; Fierro-Coronado et al., 2013Fierro-Coronado FA, Castro-Moreno MG, Ruelas-Ayala RD, Apodaca-Sánchez MÁ, Maldonado-Mendoza IE. Induced protection by Rhizophagus intraradices against Fusarium wilt of tomato. Interciencia. 2013;38:48-53.), and also act against nematodes in grapevine rootstocks (Hao et al., 2012Hao Z, Fayolle L, van Tuinen D, Chatagnier O, Li X, Gianinazzi S, Gianinazzi-Pearson V. Local and systemic mycorrhiza-induced protection against the ectoparasitic nematode Xiphinema index involves priming of defence genes response in grapevine. J Exp Bot. 2012;63:3657-72. doi:10.1093/jxb/ers046
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). Inoculation of grapevine rootstocks with AMF is therefore an option to reduce or neutralize damage from soil-borne diseases. However, the mechanisms of plant response involved in this process have not yet been completely elucidated.
One of the possibilities to increase knowledge of this interaction is the identification of proteins related to interaction between fungi and plants, especially through use of two-dimensional electrophoresis (2DE), followed by mass spectrometry (MS) (Mehta et al., 2008Mehta A, Brasileiro ACM, Souza DSL, Romano E, Campos MA, Grossi-de-Sá MF, Silva MS, Franco OL, Fragoso RR, Bevitori R, Rocha TL. Plant-pathogen interactions: what is proteomics telling us? FEBS J. 2008;275:3731-46. doi:10.1111/j.1742-4658.2008.06528.x
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). Asano et al. (2012)Asano T, Kimura M, Nishiuchi T. The defence response in Arabidopsis thaliana against Fusarium sporotrichioides. Proteome Sci. 2012;10:61. doi:10.1186/1477-5956-10-61
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found an increase in proteins related to plant defence response in Arabidopsis thaliana leaves infected with Fusarium sporotrichioides. Dumas-Gaudot et al. (2004)Dumas-Gaudot E, Valot B, Bestel-Corre G, Recorbet G, St-Arnaud M, Fontaine B, Dieu M, Raes M, Saravanan RS, Gianinnazi S. Proteomics as a way to identify extra-radicular fungal proteins from Glomus intraradices - RiT DNA carrot root mycorrhizas. FEMS Microbiol Ecol. 2004;48:401-11. doi:10.1016/j.femsec.2004.02.015
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and Recorbet et al. (2008)Recorbet G, Rogniaux H, Gianinnazi-Pearson V, Dumas-Gaudot E. Fungal proteins in the extra-radical phase of arbuscular mycorrhiza: a shotgun proteomic picture. New Phytol. 2008;181:248-60. doi:10.1111/j.1469-8137.2008.02659.x
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identified extra-radicular fungal proteins from in vitro AMF. Using 2DE analyses, Aloui et al. (2011)Aloui A, Recorbet G, Robert F, Schoefs B, Bertrand M, Henry C, Gianinazzi-Pearson V, Dumas-Gaudot E, Aschi-Smiti S. Arbuscular mycorrhizal symbiosis elicits shoot proteome changes that are modified during cadmium stress alleviation in Medicago truncatula. Plant Biol. 2011;11:75. doi:10.1186/1471-2229-11-75
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showed an up-accumulation of shoot proteins in Medicago truncatula inoculated with AMF, and Cangahuala-Inocente et al. (2011)Cangahuala-Inocente GC, Da Silva MF, Johnson JM, Manga A, van Tuinen D, Henry C, Lovato PE, Dumas-Gaudot E. Arbuscular mycorrhizal symbiosis elicits proteome responses opposite of P-starvation in SO4 grapevine rootstock upon root colonisation with two Glomus species. Mycorrhiza. 2011;21:473-93. doi:10.1007/s00572-010-0352-0
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identified 39 differentially accumulated proteins in SO4 grapevine rootstock inoculated with AMF, including proteins related to plant defence. It is thus possible to identify plant proteins differentially expressed by inoculation with mutualistic or pathogenic fungi.
We hypothesized that AMF induces plant defence response after inoculation with the pathogenic fungus FOH, and that expression of plant defence response may be influenced by grapevine rootstock resistance or susceptibility to the pathogenic fungus. The aim of this study was to identify proteins related to plant defence induced by the AMF Rhizophagus irregularis after FOH infection in two grapevine rootstocks with different levels of resistance to the pathogenic fungus.
MATERIALS AND METHODS
Experimental design, biological material and plant growth
The experiment was arranged in a 2 × 2 × 2 factorial design containing two grapevine rootstocks (SO4 and R110), two mycorrhizal treatments (inoculated or not inoculated) and two pathogenic fungus treatments (inoculated or not inoculated). The experiment, carried out in Florianópolis, southern Brazil, was repeated in time, with six replicates per treatment, consisting of a single plant each time.
SO4 and R110 rootstocks, from the plant collection of the Federal University of Santa Catarina, were micropropagated in vitro in DSD1culture medium (Lima da Silva and Doazan, 1995Lima da Silva A, Doazan JP. Une méthode d’irradiation aux rayons gamma appliquée à des porte-greffes de vigne in vitro. J Int Sci Vigne Vin. 1995;29:1-9.) containing 20 g L-1 of sucrose, 6.0 g L-1 of agar-agar and no plant growth regulators. Explants were kept between 45 and 60 days in a growth room at 25±1 °C with luminosity of 40 to 45 µmol m-2 s-1 and a 16 h d-1 photoperiod.
Inoculum of both fungi were obtained from material previously used by Dalla Costa et al. (2010)Dalla Costa M, Lovato PE, Sete PB. Micorrização e indução de quitinases e β-1,3-glucanases e resistência à fusariose em porta-enxerto de videira. Pesq Agropec Bras. 2010;45:376-83. doi:10.1590/S0100-204X2010000400005
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and kept at 4 °C until use. The AMF Rhizophagus irregularis (BEG 72, originally supplied by the French National Institute for Agriculture Research – INRA, Dijon, France) was applied as a soil-based inoculum consisting of a mixture of soil and the root system from the host plant (Allium porrum). The pathogenic fungus Fusarium oxysporum f. sp. herbemontis inoculum (CNPUV 287, originally supplied by the Brazilian Agricultural Research Corporation Centre for grape and wine - Embrapa Uva e Vinho) was kept in a sterile mixture of soil/fertilizer/sand. A sample from the FOH inoculum was added to sterile water and spread on petri dishes containing potato dextrose agar (PDA) culture medium, kept in a growth chamber for six days at 25±2 °C with luminosity of 150 µmol m-2 s-1 and 16 h d-1 photoperiod. Spores were collected by scraping the culture surface with a Drigalski spatula and adding sterile water to obtain a spore suspension with 1.0 × 107 conidia mL1.
After the in vitro growth period of the grapevine rootstocks, plants were transplanted to polystyrene trays with cells of 40 mL containing a sterile soil mixture consisting of organic substrate (Germina Plant, Turfa Fértil®), soil (Nitossolo Vermelho Distroférrico - Dystrophic Typic Hapludalf) and sand (2:1:2, v:v:v; Dalla Costa et al., 2010Dalla Costa M, Lovato PE, Sete PB. Micorrização e indução de quitinases e β-1,3-glucanases e resistência à fusariose em porta-enxerto de videira. Pesq Agropec Bras. 2010;45:376-83. doi:10.1590/S0100-204X2010000400005
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). Prior to transplant, all plants were trimmed to have 1.0-cm roots, 2.5 to 3.0 cm height and 3 to 4 leaves. For inoculation, 2.0 g of soil-based inoculum (50 to 150 spores) were placed near the plant roots, and non-inoculated plants received sterilized soil-based inoculum. Plants were kept in a growth chamber at 25± 2 °C, 98-100 % relative humidity, and luminosity of 150 µmol m-2 with a 16 h d-1 photoperiod. After 30 days, plants were transplanted into 400 mL plastic pots containing the same sterile soil mixture previously described; AMF treatments were reinoculated with 2.0 g of mycorrhizal AMF inoculum and non-inoculated plants received sterilized soil-based inoculum. The plants were grown under the same environmental conditions and watered daily with distilled water and once a week with Long Ashton nutrient solution (Smith et al., 1983Smith GS, Johnston CM, Cornforth IS. Comparison of nutrient solutions for growth of plants in sand culture. New Phytol. 1983;94:537-48. doi:10.1111/j.1469-8137.1983.tb04863.x
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) containing 10 % of the original P concentration. After 45 days, plant roots were cut to create infection points, and 1.0 mL of FOH spore suspension was placed in the soil near the roots of FOH treatments. The non-inoculated plants passed through the same procedure, with application of 1.0 mL of distilled sterile water. Subsequently, the plants were once more kept in a growth room for 14 days until harvest.
Mycorrhizal colonisation and plant growth analyses
Mycorrhizal colonization was assessed on the day the pathogenic fungus was inoculated to plants. Root pieces were taken from each plant and put in alcohol 50 °GL for storage. Mycorrhizal colonization was assessed as described by Trouvelot et al. (1986)Trouvelot A, Kough JL, Gianinazzi-Pearson V. Mesure du taux de mycorhization VA d´un système radiculaire. Recherche de méthodes d´estimation ayant une signification fonctionnelle. In: Gianinazzi-Pearson V, Gianinazzi S, editors. Mycorrhizae: physiology and genetics. Dijon: CNRS-INRA; 1986. p.217-21. after clearing and staining with trypan blue (Koske and Gemma, 1989Koske RE, Gemma JN. A modified procedure for staining roots to detect VA mycorrhizas. Mycol Res. 1989;92:486-8. doi:10.1016/S0953-7562(89)80195-9
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).
Three plants (replicates) from each treatment were harvested 14 days after pathogenic fungus inoculation. Three plants from each treatment were kept in the growth chamber for 90 more days for future evaluation of disease symptoms. At harvest, root and shoot length and fresh biomass were measured. Root samples were taken for protein analyses.
Two-dimensional electrophoresis (2DE) and mass spectrometry (MS) analysis
For protein analyses, 2.0 g (fresh biomass) of roots from each treatment were ground to a fine powder in liquid nitrogen. Phenolic protein extraction and 2DE analyses were conducted according to Cangahuala-Inocente et al. (2013)Cangahuala-Inocente GC, Amaral FP, Faleiro AC, Huergo LF, Arisi ACM. Identification of six differentially accumulated proteins of Zea mays seedlings (DKB240 variety) inoculated with Azospirillum brasilense strain FP2. Eur J Soil Biol. 2013;58:45-50. doi:10.1016/j.ejsobi.2013.06.002
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. Protein concentrations were estimated according to Bradford (1976)Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54. doi:10.1016/0003-2697(76)90527-3
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and modified by Ramagli and Rodrigues (1985)Ramagli LS, Rodriguez LV. Quantification of microgram amounts of protein in two dimensional polyacrylamide gel electrophoresis sample buffers. Electrophoresis. 1985;6:559-63. doi:10.1002/elps.1150061109
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. A protein concentration of 500 μg from each sample was subjected to isoelectric focusing (first dimension) in an immobilized linear pH gradient 3-10 on 13 cm strips (IPG strips - GE Healthcare) on an Ettan-IPGPhor instrument (GE Healthcare) with a total of 35,000 Vh. After this step, the strips were equilibrated for 15 min in equilibration buffer (50 mmol L-1, pH 8.8 Tris-HCl, 6 mol L-1 urea, 30 % (v/v) glycerol, 2 % (w/v) SDS and 0.002 % (w/v) bromophenol blue), containing 1 % (w/v) DTT and 2.5 % (w/v) iodoacetomide. The strips were attached to the top of 12 % polyacrylamide gels in a Hoefer SE 600 Ruby System (GE Healthcare) and run at 4 °C and 10 mA for 1 h, 20 mA for 1 h and 30 mA for 4 h (second dimension). The gels were stained with Coomassie Brillant Blue G250 solution according to Mathesius et al. (2001)Mathesius U, Keijzers G, Natera SHA, Weinman JJ, Djordjevic MA, Rolfe BG. Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting. Proteomics. 2001;1:1424-40. doi:10.1002/1615-9861(200111)1:11<1424::AID-PROT1424>3.0.CO;2-J
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. Stained gels were scanned in an Image Scanner (GE Healthcare) and analysed with ImageMaster 2D Platinum 7.0 (GE Healthcare). The protein spots were identified and selected based on comparative analyses of the gels from non-inoculated and inoculated treatments (p≤0.05). Proteins were identified as unique, absent or up- or down-accumulated by AMF R. irregularis and/or by the pathogenic fungus F. oxysporum f. sp. herbemontis. Three gels were obtained from each treatment and biological replicate, resulting in a total of six gels per treatment.
The selected proteins spots from 2DE gels were manually cut, destained with discoloration solution (50 % acetonitrile, 25 mmol L-1 ammonium bicarbonate), dried under a vacuum with pure acetonitrile and digested with porcine trypsin. The peptides were recovered with extraction solution (50 % acetonitrile, 5 % TFA), concentrated, dried, solubilized in 0.1 % trifluoroacetic acid and analysed in a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) AUTOFLEX II TOF-TOF instrument (Burker Daltonics). One µL of recrystallized HCCA matrix (Burker Daltonics) was dissolved (0.1 % trifluoracetic acid and 70 % acetonitrile) and spotted once on top of samples (Shevchenko et al., 1996Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem. 1996;68:850-8. doi:10.1021/ac950914h
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). The MALDI-TOF mass spectra were acquired in a mass range of 1,000 Da to 3,200 Da. Protein identification was performed by the Mascot software database (http://www.matrixscience.com/) and subsequently by the NCBInr database to identify homologous proteins (http://www.ncbi.nlm.nih.gov/). The following parameters were used for the database search: fixed modifications of carbamidomethylation cysteine, taxonomy of Viridiplantae and trypsin as an enzyme. The molecular functions of proteins were confirmed on the UniprotKB database (http://www.uniprot.org/).
Evaluation of disease symptoms
The evaluation of disease symptoms caused by F. oxysporum f. sp. herbemontis was performed 90 days after inoculation with FOH, following the disease index method of Grigoletti Júnior (1985)Grigoletti Júnior A. Fusariose da videira: caracterização, variabilidade do Fusarium oxysporum f. sp. herbemontis e fontes de resistência em Vitis spp. [tese]. Viçosa, MG: Universidade Federal de Viçosa; 1985.. Plants with an external and internal degree of infection between 1 and 2 are resistant to FOH; plants exhibiting degree 3 are tolerant; plants with a degree between 4 and 5 are susceptible to FOH.
Statistics
A factorial analysis of variance was performed for growth parameters, mycorrhizal colonisation, protein concentration and pathogenic symptoms. Mean separation of treatments with significance was done with specific post-hoc comparisons using the Student Newman Keuls method (SNK) at a significance level of 5 %. Statistical analyses were carried out using R version 3.2.1 software (The R Foundation, 2015The R Foundation. The R Project for Statistical Computing [internet]. Vienna: The R foundation; 2015 [accessed on: 15 June 2015]. Available at: http://www.r-project.org/.
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).
RESULTS AND DISCUSSIONS
Mycorrhizal colonisation, plant growth, and disease symptoms
Arbuscular mycorrhizal fungi-inoculated grapevine rootstocks had a 100 % frequency of fungal infection and over 50 % intensity of root cortex colonisation, with no significant differences between SO4 and R110 rootstocks. In addition to high colonisation intensity, roots had arbuscules (over 50 % frequency), indicating mycorrhizal functionality. Shoot and root biomasses were significantly higher in both grapevine rootstocks inoculated with AMF in comparison to non-inoculated plants (Table 1). However, there were no differences between rootstocks in plant growth, and root length in non-inoculated rootstock was higher than in all other treatments. It has been shown that mycorrhizal plants may have fewer and shorter roots than non-mycorrhizal plants, since extraradicular AMF mycelium increases the volume of soil explored by the plant root system, allowing higher water and nutrient uptake (Locatelli et al., 2002Locatelli LM, Vitovski CA, Lovato PE. Sistema radicular de porta-enxertos micropropagados de macieira inoculados com fungos micorrízicos arbusculares. Pesq Agropec Bras. 2002;37:1239-45.). Therefore, these results confirm the ability of AMF to benefit plant growth, and the increase in shoot and root biomass is consistent with previous studies with grapevine rootstocks (Dalla Costa et al., 2010Dalla Costa M, Lovato PE, Sete PB. Micorrização e indução de quitinases e β-1,3-glucanases e resistência à fusariose em porta-enxerto de videira. Pesq Agropec Bras. 2010;45:376-83. doi:10.1590/S0100-204X2010000400005
https://doi.org/10.1590/S0100-204X201000...
; Anzanello et al., 2011Anzanello R, Souza PVD, Casamali B. Fungos micorrízicos arbusculares (FMA) em porta-enxertos micropropagados de videira. Bragantia. 2011;70:409-15. doi:10.1590/S0006-87052011000200023
https://doi.org/10.1590/S0006-8705201100...
; Cangahuala-Inocente et al., 2011Cangahuala-Inocente GC, Da Silva MF, Johnson JM, Manga A, van Tuinen D, Henry C, Lovato PE, Dumas-Gaudot E. Arbuscular mycorrhizal symbiosis elicits proteome responses opposite of P-starvation in SO4 grapevine rootstock upon root colonisation with two Glomus species. Mycorrhiza. 2011;21:473-93. doi:10.1007/s00572-010-0352-0
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) or with other plant species (Lovato et al., 2006Lovato PE; Trouvelot A, Gianinazzi-Pearson V; Gianinazzi S. Enhanced growth of wild cherry using micropropagated plants and mycorrhizal inoculation. Agron Sustain Dev. 2006;26:209-13.; Farzaneh et al., 2011Farzaneh M, Vierheilig H, Lössl A, Kaul HP. Arbuscular mycorrhiza enhances nutrient uptake in chickpea. Plant Soil Environ. 2011;57:465-70.; Cavagnaro et al., 2012Cavagnaro TR, Barrios-Masias FH, Jackson LE. Arbuscular mycorrhizas and their role in plant growth, nitrogen interception and soil gas efflux in an organic production system. Plant Soil. 2012;353:181-94. doi:10.1007/s11104-011-1021-6
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; Evelin et al., 2012Evelin H, Giri B, Kapoor R. Contribution of Glomus intraradices inoculation to nutrient acquisition and mitigation of ionic imbalance in NaCl-stressed Trigonella foenum-graecum. Mycorrhiza. 2012;22:203-17. doi:10.1007/s00572-011-0392-0
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; Mohandas, 2012Mohandas S. Arbuscular mycorrhizal fungi benefit mango (Mangifera indica L.) plant growth in the field. Sci Hortic. 2012;143:43-8. doi:10.1016/j.scienta.2012.05.030
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; Steinkellner et al., 2012Steinkellner S, Hage-Ahmed K, García-Garrido JM, Illana A, Ocampo JA, Vierheilig H. A comparison of wild-type, old and modern tomato cultivars in the interaction with arbuscular mycorrhizal fungus Glomus mosseae and the tomato pathogen Fusarium oxysporum f. sp. lycopersici. Mycorrhiza. 2012;22:189-94. doi:10.1007/s00572-011-0393-z
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). Anzanello et al. (2011)Anzanello R, Souza PVD, Casamali B. Fungos micorrízicos arbusculares (FMA) em porta-enxertos micropropagados de videira. Bragantia. 2011;70:409-15. doi:10.1590/S0006-87052011000200023
https://doi.org/10.1590/S0006-8705201100...
detected an increase in shoot and root biomass in SO4 and P1103 rootstocks inoculated with AMF, and Dalla Costa et al. (2010)Dalla Costa M, Lovato PE, Sete PB. Micorrização e indução de quitinases e β-1,3-glucanases e resistência à fusariose em porta-enxerto de videira. Pesq Agropec Bras. 2010;45:376-83. doi:10.1590/S0100-204X2010000400005
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also showed an increase in shoot biomass in SO4 rootstock inoculated with AMF. In a study using different varieties of tomatoes inoculated and not inoculated with AMF and inoculated and not inoculated with F. oxysporum f. sp. lycopersici, Steinkellner et al. (2012)Steinkellner S, Hage-Ahmed K, García-Garrido JM, Illana A, Ocampo JA, Vierheilig H. A comparison of wild-type, old and modern tomato cultivars in the interaction with arbuscular mycorrhizal fungus Glomus mosseae and the tomato pathogen Fusarium oxysporum f. sp. lycopersici. Mycorrhiza. 2012;22:189-94. doi:10.1007/s00572-011-0393-z
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detected a positive effect of AMF in plant biomass in some tomato varieties inoculated only with AMF and also when co-inoculated with both fungi.
Root, shoot and total fresh weight, and root length in grapevine rootstocks inoculated (Myc) and not inoculated (NM) with the mycorrhizal fungus Rhizophagus irregularis and inoculated (+FOH) or not inoculated (-FOH) with the pathogenic fungus Fusarium oxysporum f. sp. herbemontis. Mean for two rootstocks (SO4 and R110)
Disease symptoms were less marked in grapevine rootstocks inoculated with both fungi than in plants inoculated only with FOH (Table 2). That suggests a bioprotective effect of AMF against FOH colonization in both grapevine rootstocks, which is more marked in the susceptible SO4 rootstock. Dalla Costa et al. (2010)Dalla Costa M, Lovato PE, Sete PB. Micorrização e indução de quitinases e β-1,3-glucanases e resistência à fusariose em porta-enxerto de videira. Pesq Agropec Bras. 2010;45:376-83. doi:10.1590/S0100-204X2010000400005
https://doi.org/10.1590/S0100-204X201000...
also detected a decrease in disease symptoms in SO4 grapevine rootstocks co-inoculated with AMF and FOH. In two independent studies, Steinkellner et al. (2012)Steinkellner S, Hage-Ahmed K, García-Garrido JM, Illana A, Ocampo JA, Vierheilig H. A comparison of wild-type, old and modern tomato cultivars in the interaction with arbuscular mycorrhizal fungus Glomus mosseae and the tomato pathogen Fusarium oxysporum f. sp. lycopersici. Mycorrhiza. 2012;22:189-94. doi:10.1007/s00572-011-0393-z
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and Fierro-Coronado et al. (2013)Fierro-Coronado FA, Castro-Moreno MG, Ruelas-Ayala RD, Apodaca-Sánchez MÁ, Maldonado-Mendoza IE. Induced protection by Rhizophagus intraradices against Fusarium wilt of tomato. Interciencia. 2013;38:48-53. showed that tomato plants co-inoculated with AMF and Fusarium oxysporum f. sp. lycopersici had lower disease infection than plants inoculated only with the pathogenic fungus, indicating the bioprotective effect of AMF against that fungus. This bioprotective effect has been linked to a stimulation of plant defence mechanisms (Schenkluhn et al., 2010Schenkluhn L, Hohnjec N, Niehaus K, Schmitz U, Colditz B. Differential gel electrophoresis (DIGE) to quantitatively monitor early symbiosis- and pathogenesis-induced changes of the Medicago truncatula root proteome. J. Proteom. 2010;73:453-768. doi:10.1016/j.jprot.2009.10.009
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), which occur when either mutualistic or pathogenic microorganisms are inoculated. Identification of defence-related proteins may provide clues about the multiple mechanisms that may be involved.
Two-dimensional electrophoresis and mass spectrometry analysis
Total protein contents were higher in R110 grapevine rootstocks than in SO4, due to increases associated with AMF and FOH inoculation (Table 3). Simultaneously, 2DE protein gels from both grapevine rootstocks showed differences in their protein profiles (Figure 1). The R110 grapevine rootstock yielded 73 differentially accumulated proteins, whereas 59 differentially accumulated proteins were found in the SO4 rootstock (Table 4). AMF and FOH inoculation, and also co-inoculation with both fungi, resulted in differences in protein accumulation. According to Anzanello et al. (2011)Anzanello R, Souza PVD, Casamali B. Fungos micorrízicos arbusculares (FMA) em porta-enxertos micropropagados de videira. Bragantia. 2011;70:409-15. doi:10.1590/S0006-87052011000200023
https://doi.org/10.1590/S0006-8705201100...
, benefits of AMF depend on interaction between the fungus and the rootstock, which might explain the differences in protein profile. Concomitantly, differences in the protein profile may be related to the different levels of resistance from the grapevine rootstocks to FOH, also affecting the interaction between plant and fungi. Xu et al. (2015)Xu PL, Liu YX, Qin HY, Ai J, Fan ST, Yang YM, Zhao Y, Li XY, Li XH. Proteomic analysis of the resistant responses of two Vitis amurensis cultivars to Plasmopara viticola infections. Curr Proteomics. 2015;12:63-8. doi:10.2174/157016461201150506202735
https://doi.org/10.2174/1570164612011505...
also showed differences in the proteome of two cultivars of Vitis amurensis with different levels of resistance to a pathogenic fungus.
Protein concentration in SO4 and R110 grapevine rootstock roots inoculated (Myc) or not inoculated (NM) with the mycorrhizal fungus Rhizophagus irregularis and inoculated (+FOH) or not inoculated (-FOH) with the pathogenic fungus Fusarium oxysporum f. sp. herbemontis, 60 days after transplanting and the second inoculation with mycorrhizal fungus
Representative image of 2DE gel showing the differentially accumulated proteins (red circles) between the SO4 and R110 grapevine rootstocks and between the control treatment (NM -FOH) and co-inoculation with the mycorrhizal fungus Rhizophagus irregularis and the pathogenic fungus Fusarium oxysporum f. sp. herbemontis (Myc +FOH).
Differentially accumulated proteins from the root system of two grapevine rootstocks (SO4 and R110) inoculated or not inoculated with the arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis and inoculated or not inoculated with the pathogenic fungus Fusarium oxysporum f. sp. herbemontis (FOH), 60 days after transplanting and second inoculation with mycorrhizal fungus
Mass spectrometry analysis revealed nine proteins related to defence response in SO4 grapevine rootstock (Table 5). Among them, two proteins (ID 252 and 256) were induced by AMF and also by FOH inoculation, and three proteins (ID 629, 655 and 670) were induced by co-inoculation with AMF and FOH. Meanwhile, four proteins (ID 2099, 2101, 2012 and 2143) were down-accumulated in all treatments, in comparison to the non-inoculated treatment. In general, those proteins were up-accumulated in response to AMF inoculation as well as to FOH inoculation. Plant colonisation by AMF also induces activation of the plant defence system, which is, however, readily suppressed, due to recognition of mutualistic symbiosis by the plant. Concomitantly, plants inoculated with AMF are capable of rapidly reactivating their defence system (priming) when facing a stress situation, resulting in enhanced resistance (Pozo and Azcón-Aguilar, 2007Pozo MJ, Azcón-Aguilar C. Unravelling mycorrhiza-induced resistance. Curr Opin Plant Biol. 2007;10:393-8. doi:10.1016/j.pbi.2007.05.004
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; Dalla Costa et al., 2010Dalla Costa M, Lovato PE, Sete PB. Micorrização e indução de quitinases e β-1,3-glucanases e resistência à fusariose em porta-enxerto de videira. Pesq Agropec Bras. 2010;45:376-83. doi:10.1590/S0100-204X2010000400005
https://doi.org/10.1590/S0100-204X201000...
). Therefore, in the SO4 grapevine rootstocks co-inoculated with both fungi, AMF acted as a bioprotective agent against FOH.
Spot number identification from gel (Spot ID), protein coverage in % of protein length, accession number from database, experimental and theoretical isoelectric point (Exptl/theor pI), experimental and theoretical molecular weight (Exptl/theor MW) and protein name from database of SO4 and R110 grapevine rootstock roots inoculated or not inoculated with the mycorrhizal fungus Rhizophagus irregularis and inoculated or not inoculated with the pathogenic fungus Fusarium oxysporum f. sp. herbemontis
The spot ID 252, “serine/threonine protein kinase A”, belongs to a class of plant resistance (R-) gene products that determine recognition of pathogen infection and trigger the plant defence system (Di Gaspero and Cipriani, 2003Di Gaspero G, Cipriani EG. Nucleotide binding site/leucine-rich repeats, Pto-like and receptor-like kinases related to disease resistance in grapevine. Mol Gen Genomics. 2003;269:612-23. doi:10.1007/s00438-003-0884-5
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). Two cultivars of Vitis amurensis had the expression of R-genes enhanced in response to pathogenic fungus infection (Xu et al., 2015Xu PL, Liu YX, Qin HY, Ai J, Fan ST, Yang YM, Zhao Y, Li XY, Li XH. Proteomic analysis of the resistant responses of two Vitis amurensis cultivars to Plasmopara viticola infections. Curr Proteomics. 2015;12:63-8. doi:10.2174/157016461201150506202735
https://doi.org/10.2174/1570164612011505...
), and a similar process was found in the present study with AMF inoculation. The spot ID 256 is a pathogenesis-related protein 1 (PR-1 protein), stimulated by plant stress as a defence response (Edreva, 2005Edreva A. Pathogenesis-related proteins: research progress in the last 15 years. Gen Appl Plant Physiol. 2005;31:105-24.). The pathogenesis-related proteins (PR) are synthetized by numerous plants as a defence mechanism against various pathological processes, and these proteins are classified in different groups (Xu et al., 2015Xu PL, Liu YX, Qin HY, Ai J, Fan ST, Yang YM, Zhao Y, Li XY, Li XH. Proteomic analysis of the resistant responses of two Vitis amurensis cultivars to Plasmopara viticola infections. Curr Proteomics. 2015;12:63-8. doi:10.2174/157016461201150506202735
https://doi.org/10.2174/1570164612011505...
). The spot ID 629 is a cysteine-rich thionin protein, an antimicrobial peptide toxic to several biological systems, belonging to a group of proteins which include the pathogenesis related protein 13 (PR-13) (Nawrot et al., 2014Nawrot R, Barylski J, Nowicki G, Broniarczyk J, Buchwald W, Goździcka-Józefiak A. Plant antimicrobial peptides. Folia Microbiol. 2014;59:181-96. doi:10.1007/s12223-013-0280-4
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). Epple et al. (1995)Epple P, Apel K, Bohlmann H. An Arabidopsis thaliana thionin gene is inducible via a signal transduction pathway different from that for pathogenesis-related proteins. Plant Physiol. 1995;109:813-20. doi:10.1104/pp.109.3.813
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detected an increase in thionin concentration in seeds inoculated with F. oxysporum f. sp. matthiolae. The spot ID 2102 is also a pathogenesis-related protein belonging to group 2 (PR-2). The enzyme β-1,3-glucanase, a PR-2 protein, catalyses the pathogen cell wall polysaccharide β-1,3-glucan, thus preventing fungal diseases (Edreva, 2005Edreva A. Pathogenesis-related proteins: research progress in the last 15 years. Gen Appl Plant Physiol. 2005;31:105-24.). This enzyme has been detected in grapevine rootstocks challenged by F. oxysporum f. sp. herbemontis (Dalla Costa et al., 2010Dalla Costa M, Lovato PE, Sete PB. Micorrização e indução de quitinases e β-1,3-glucanases e resistência à fusariose em porta-enxerto de videira. Pesq Agropec Bras. 2010;45:376-83. doi:10.1590/S0100-204X2010000400005
https://doi.org/10.1590/S0100-204X201000...
). Maxson-Stein et al. (2002)Maxson-Stein K, He SY, Hammerschmidt R, Jones AL. Effect of treating apple trees with acibenzolar-S-methyl on fire blight and expression of pathogenesis-related protein genes. Plant Dis. 2002;86:785-90. doi:10.1094/PDIS.2002.86.7.785
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detected PR-2 proteins in apple trees inoculated with a pathogenic fungus and sprayed with acibenzolar-S-methyl, leading to a decrease in pathogenesis symptoms. In summary, all these proteins are related to plant defence, and had their production enhanced by AMF inoculation.
Arbuscular mycorrhizal fungi (AMF) inoculation also affected other proteins, less directly linked to plant defence against pathogens. The spot ID 655, caffeic acid-O-methyltransferase, takes part in the lignin biosynthesis pathway, converting caffeic acid to ferulic acid during the phenylpropanoid pathway (Zhang et al., 2011Zhang YM, Zhao JM, Xiang Y, Bian XC, Zuo QM, Shen Q, Gai JY, Xing H. Proteomics study of changes in soybean lines resistant and sensitive to Phytophthora sojae. Proteome Sci. 2011;9:52. doi:10.1186/1477-5956-9-52
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). Plant cell wall lignification may also be triggered by plant defence mechanisms, preventing enzymatic hydrolysis and plant penetration by pathogenic fungi (Barros et al., 2015Barros J, Serk H, Granlund I, Pesquet E. The cell biology of lignification in higher plants. Ann Bot. 2015;115:1053-74. doi:10.1093/aob/mcv046
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). The spot ID 670 is an integral membrane protein called SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) that mediates vesicle fusion between the organelles (Sanderfoot and Raikhel, 1999Sanderfoot AA, Raikhel NV. The specificity of vesicle trafficking: coat proteins and SNAREs. Plant Cell. 1999;11:629-41. doi:10.1105/tpc.11.4.629
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). SNARE proteins are located on the surfaces of vesicles and target membranes, and their main role is to identify the target organelle and mediate the fusion of vesicles with the organelle. Collins et al. (2003)Collins NC, Thordal-Christensen H, Lipka V, Bau S, Kombrink E, Qiu JL, Hückelhoven R, Stein M, Freialdenhoven A, Somerville SC, Schulze-Lefert P. SNARE-protein-mediated disease resistance at the plant cell wall. Nature. 2003;425:973-7. doi:10.1038/nature02076
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identified that the SNARE complex mediates the expression of plant resistance genes against pathogenic fungus penetration. The spot ID 2099 is an actin, the main component of the plant cytoskeleton, contributing to cell maintenance. Actin may also act as a defence mechanism in the plant hypersensitive cell-death reaction against pathogenic infection. Chang et al. (2011)Chang X, Heene E, Qiao F, Nick P. The phytoalexin resveratrol regulates the initiation of hypersensitive cell death in Vitis cell. PLoS ONE. 2011;6:10. doi:10.1371/journal.pone.0026405
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showed an increase in actin bundling and ensuing cell death in grapevines treated with phytoalexin. The spot ID 2143 corresponds to a superfamily of related proteins called Ras, constituted by small GTPase proteins. Their main action is on the signal transduction pathway coupling receptors to intracellular signals required for cell growth stimulation and differentiation, and also for actin polymerization (Hall, 1993Hall A. Ras-related proteins. Curr Opin Cell Biol. 1993;5:265-8. doi:10.1016/0955-0674(93)90114-6
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). Therefore, accumulation of this protein could be directly related to accumulation of actin, as described above (spot ID 2099). The spot ID 2101 is an antiviral ribosome inactivating protein (Rip) related also to plant defence response against fungi. This protein is a toxin that inactivates the ribosome by cleavage of N-glycosidase at rRNA adenine, with consequent protein synthesis interruption, leading to cell death (Schrot et al., 2015Schrot J, Weng A, Melzig MF. Ribosome-Inactivating and related proteins. Toxins. 2015;7:1556-615. doi:10.3390/toxins7051556
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).
Mass spectrometry of R110 grapevine rootstock extract revealed a smaller number of proteins related to plant defence response (Table 5). Among those six proteins, one of them (ID 1223) was down-accumulated in all treatments in comparison to non-inoculated plants. Four proteins (ID 1370, 1447, 1495 and 2077) were present in AMF-inoculated plants and also in plants with co-inoculation of AMF and FOH. Therefore, these four proteins were accumulated due to inoculation with AMF, since they were not detected in plants inoculated only with FOH. This suggests that in the co-inoculation treatment, the AMF acted as a bioprotective agent against inoculation with FOH, priming the plant defence response. Meanwhile, only one protein (ID1276) was induced by FOH inoculation and also by co-inoculation with AMF and FOH. In this case, this protein is only related to FOH inoculation.
The spot ID 1223 is a cinnamate 4-hydroxylase acting on electron transport and oxidoreductase activity. This enzyme catalyses the second step of the phenylpropanoid pathway, in which a wide range of plant defence response compounds are synthesized (Chen et al., 2014Chen L, Guo H, Lin Y, Wu Y, Cheng H. Molecular cloning and characterization of the cinnamate 4-hydroxylase gene from Eupatorium adenophorum. Weed Biol Manage. 2014;14:167-77. doi:10.1111/wbm.12044
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). The spot ID 1370 is an enzyme complex, 20S proteasome beta subunit, a component of the 26S proteasome. The proteasome degrades undesired or damaged proteins in the organism. Proteins are recognized by the proteasome via the signal molecule ubiquitin, attached to the proteins during the ubiquitination process, thus starting the ubiquitin proteasome pathway. This pathway contributes to cell proliferation, hormone responses, development, senescence and resistance to abiotic and biotic stress (Marques et al., 2009Marques AJ, Palanimurugan R, Matias AC, Ramos PC, Dohmen RJ. Catalytic mechanism and assembly of the proteasome. Chem Rev. 2009;109:1509-36. doi:10.1021/cr8004857
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). The spot ID 1276 is a SKP1 protein acting on ubiquitination, and consequently also involved in the ubiquitin proteasome pathway (Zheng et al., 2002Zheng N, Schulman BA, Song L, Miller JJ, Jeffrey PD, Wang P, Chu C, Koepp DM, Elledge SJ, Pagano M, Conaway RC, Conaway JW, Harper JW, Pavletich NP. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature. 2002;416:703-9. doi:10.1038/416703a
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). Therefore, accumulation of this protein is directly related to spot ID 1370, and both act in plant defence response. Cangahuala-Inocente et al. (2011)Cangahuala-Inocente GC, Da Silva MF, Johnson JM, Manga A, van Tuinen D, Henry C, Lovato PE, Dumas-Gaudot E. Arbuscular mycorrhizal symbiosis elicits proteome responses opposite of P-starvation in SO4 grapevine rootstock upon root colonisation with two Glomus species. Mycorrhiza. 2011;21:473-93. doi:10.1007/s00572-010-0352-0
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showed up-accumulation of two proteins related to the proteasome complex and also accumulation of ubiquitin in roots of SO4 grapevine rootstocks inoculated with the same AMF used in this study. Margaria and Palmano (2011)Margaria P, Palmano S. Response of the Vitis vinifera L. cv. ‘Nebbiolo’ proteome to Flavescence dorée phytoplasma infection. Proteomics. 2011;11:212-24. doi:10.1002/pmic.201000409
https://doi.org/10.1002/pmic.201000409...
also showed up-accumulation of two proteins from the proteasome complex in Vitis vinifera (Nebbiolo) inoculated with phytoplasma. The accumulation of these two proteins in the R110 grapevine rootstock as well suggests a direct relation between the ubiquitin proteasome pathway and plant defence response by grapevine plants. The spot ID 1447 is a transcription factor called heat-shock factor binding protein. Under stress conditions, the heat shock factor induces the expression of heat shock proteins involved in plant defence response (Asthir, 2015Asthir B. Mechanisms of heat tolerance in crop plants. Biol Plant. 2015;59:620-8. doi:10.1007/s10535-015-0539-5
https://doi.org/10.1007/s10535-015-0539-...
). Mycorrhizal grapevine roots upregulated the expression of heat-shock proteins, due to inoculation with an ectoparasitic nematode (Hao et al., 2012Hao Z, Fayolle L, van Tuinen D, Chatagnier O, Li X, Gianinazzi S, Gianinazzi-Pearson V. Local and systemic mycorrhiza-induced protection against the ectoparasitic nematode Xiphinema index involves priming of defence genes response in grapevine. J Exp Bot. 2012;63:3657-72. doi:10.1093/jxb/ers046
https://doi.org/10.1093/jxb/ers046...
). The spot ID 1495 was identified in the database with the molecular function of S-adenosyl-methionine-dependent methyltransferase activity. This protein catalyses the transfer of methyl groups S-adenosyl-methionine to other molecules (methylation) and is involved in biosynthesis of a wide variety of metabolites involved in plant defence, growth, and development (Joshi and Chiang, 1998Joshi CP, Chiang VL. Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases. Plant Mol Biol. 1998;37:663-74. doi:10.1023/A:1006035210889
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). The biological process of the spot ID 2077 is described as plant defence response. Xu et al. (2011)Xu MR, Cruz CMV, Fu BY, Zhu LH, Zhou YL, Li ZK. Different partners of gene expression in rice varieties undergoing a resistant or susceptible interaction with the bacterial leaf streak pathogen. Afr J Biotechnol. 2011;10:14419-38. doi:10.5897/AJB11.1317
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described this protein as pathogenesis-related protein 10 (PR-10) and detected its increase in rice plants inoculated with the pathogenic fungus Xanthomonas oryzae pv. oryzicola, which damages rice shoots. Hao et al. (2012)Hao Z, Fayolle L, van Tuinen D, Chatagnier O, Li X, Gianinazzi S, Gianinazzi-Pearson V. Local and systemic mycorrhiza-induced protection against the ectoparasitic nematode Xiphinema index involves priming of defence genes response in grapevine. J Exp Bot. 2012;63:3657-72. doi:10.1093/jxb/ers046
https://doi.org/10.1093/jxb/ers046...
detected upregulation of the PR-10 gene during AMF-induced bioprotection against nematode inoculation in grapevine roots, suggesting the priming effect of AMF to plant defence response, as observed in the present study in roots challenged by a fungus.
In summary, we detected a range of defence-related proteins that were expressed or upregulated in both grapevine rootstocks associated with either mutualistic or pathogenic fungi. There were a larger number of defence-related proteins in the susceptible SO4 rootstock than in the less susceptible 110 rootstock, suggesting that resistant or tolerant plants may block the infection process at several stages, resulting in less severe effects of endophytic fungi on protein expression in the root system. The results presented here show that the presence of the mutualistic and the pathogenic fungi results in a highly complex process, with a series of steps that must be taken into account in plant breeding and management.
CONCLUSIONS
The AMF Rhizophagus irregularis benefits grapevine rootstocks by improving plant growth and reducing damages caused by the pathogenic fungus Fusarium oxysporum f. sp. herbemontis.
Root protein profiles of grapevine rootstocks are dependent on their level of pathogen resistance.
Accumulation of proteins related to plant defence response in grapevine rootstocks varies with the combination of the rootstock, pathogen fungus, and arbuscular mycorrhizal fungus.
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Publication Dates
-
Publication in this collection
2017
History
-
Received
1 Mar 2016 -
Accepted
15 June 2016