Elemental composition of strawberry plants inoculated with the plant growth‐promoting bacterium Azospirillum brasilense REC3, assessed with scanning electron microscopy and energy dispersive X‐ray analysis

The elemental composition of strawberry plants (Fragaria ananassa cv. Macarena) inoculated with the plant growth‐promoting bacterium Azospirillum brasilense REC3, and non‐inoculated controls, was studied using scanning electron microscopy (SEM) and energy dispersive X‐ray (EDS) analysis. This allowed simultaneous semi‐quantification of different elements in a small, solid sample. Plants were inoculated and grown hydroponically in 50% or 100% Hoagland solution, corresponding to limited or optimum nutrient medium, respectively. Bacteria‐inoculated plants increased the growth index 45% and 80% compared to controls when grown in 100% and 50% Hoagland solution, respectively. Thus, inoculation with A. brasilense REC3 in a nutrient‐limited medium had the strongest effect in terms of increasing both shoot and root biomass and growth index, as already described for Azospirillum inoculated into nutrient‐poor soils. SEM‐EDS spectra and maps showed the elemental composition and relative distribution of nutrients in strawberry tissues. Leaves contained C, O, N, Na, P, K, Ca and Cu, while roots also had Si and Cl. The organic fraction (C, O and N) accounted for over 96.3% of the total chemical composition; of the mineral fraction, Na had higher accumulation in both leaves and roots. Azospirillum‐inoculated and control plants had similar elemental quantities; however, in bacteria‐inoculated roots, P was significantly increased (34.33%), which constitutes a major benefit for plant nutrition, while Cu content decreased (35.16%).     

Role of ethylene and related gene expression in the interaction between strawberry plants and the plant growth‐promoting bacterium Azospirillum brasilense

• Induced systemic resistance (ISR) is one of the indirect mechanisms of growth promotion exerted by plant growth‐promoting bacteria, and can be mediated by ethylene (ET). We assessed ET production and the expression of related genes in the Azospirillum–strawberry plant interaction.
• Ethylene production was evaluated by gas chromatography in plants inoculated or not with A. brasilense REC3. Also, plants were treated with AgNO₃, an inhibitor of ET biosynthesis; with 1‐aminocyclopropane‐1‐carboxylic acid (ACC), a precursor of ET biosynthesis; and with indole acetic acid (IAA). Plant dry biomass and the growth index were determined to assess the growth‐promoting effect of A. brasilense REC3 in strawberry plants. Quantitative real time PCR (qRT‐PCR) was performed to analyse relative expression of the genes Faetr1, Faers1 and Faein4, which encode ET receptors; Factr1 and Faein2, involved in the ET signalling pathway; Faacs1 encoding ACC synthase; Faaco1 encoding ACC oxidase; and Faaux1 and Faami1 for IAA synthesis enzymes.
• Results showed that ET acts as a rapid and transient signal in the first 12 h post‐treatment. A. brasilense REC3‐inoculated plants had a significantly higher growth index compared to control plants. Modulation of the genes Faetr1, Faers1, Faein4, Factr1, Faein2 and Faaco1 indicated activation of ET synthesis and signalling pathways. The up‐regulation of Faaux1 and Faami1 involved in IAA synthesis suggested that inoculation with A. brasilense REC3 induces production of this auxin, modulating ET signalling.
• Ethylene production and up‐regulation of genes associated with ET signalling in strawberry plants inoculated with A. brasilense REC3 support the priming activation characteristic of ISR. This type of resistance and the activation of systemic acquired resistance previously observed in this interaction indicate that both are present in strawberry plants, could act synergistically and increase protection against pathogens.

     

The Polar Flagellin of Azospirillum brasilense REC3 Induces a Defense Response in Strawberry Plants Against the Fungus Macrophomina phaseolina

Microbe-associated molecular patterns (MAMPs) are conserved molecules able to trigger plant resistance. The aim of this work was to evaluate the capacity of Azospirillum brasilense REC3 polar flagellin AzFlap as a MAMP, eliciting biochemical, histological, and molecular defense responses that can provide strawberry plants protection against the pathogenic fungus Macrophomina phaseolina. Strawberry plants were treated with AzFlap on leaves or with the isolate REC3 on leaves or roots. Salicylic acid content, biofilm formation, callose and lignin depositions, stomatal closure, ROS, and the expression of defense-related genes such as FaPR1, FaCAT, FaRBOH-D, FaRBOH-F, FaCHI23, FaCHI2-2, and FaGSL5 were evaluated. Phytopathogenic assays in plants treated with AzFlap or REC3 and infected with M. phaseolina were also performed. Results showed that plants leaf treated with AzFlap or root treated with REC3 caused the accumulation of ROS, salicylic acid, callose, lignin, the increase of biofilm formation on leaves, and stomatal closure. The evaluation of the expression of genes associated to defense response indicated the activation of the innate immunity of strawberry plants. The level of gene expression was strongly time and treatment dependent, suggesting a complex regulation of defense signaling. Root inoculations with REC3 or foliar treatment with AzFlap were able to reduce plant mortality, showing the effectiveness of both treatments to control M. phaseolina. These results indicate that flagellin AzFlap from A. brasilense REC3 behaves as a MAMP that activates a defense response against M. phaseolina in strawberry plants.

     

Bacillus methylotrophicus M4-96 Stimulates the Growth of Strawberry (Fragaria × ananassa ‘Aromas’) Plants In Vitro and Slows Botrytis cinerea Infection by Two Different Methods of Interaction

Bacillus methylotrophicus M4-96 is a beneficial rhizobacterium that has been isolated from the rhizosphere of maize (Zea mays). In this study, we investigated its efficacy as a plant growth promoter for strawberry in vitro, as well as its ability to induce callose deposition in leaves to reduce the severity of Botrytis cinerea infection. Two methods of plant-bacterial interaction were used: inoculation near the root and emission of volatile compounds with no physical contact. Plant biomass increased under both treatments, but with developmental parameters of the plants differentially stimulated by each method. Root inoculation increased petiole number and root length, whereas bacterial volatiles increased petiole length and root number. A chemical analysis of the bacterial culture revealed the presence of indole acetic acid (0.21 μg L⁻¹) and gibberellic acid (6.16 μg L⁻¹). Acetoin was previously identified as the major volatile produced by the bacteria, and its application to strawberry explants increased their growth and development. Furthermore, when acetoin and both phytoregulators were added to the culture media, these positive effects were enhanced. The inoculation method also affected the size and quantity of callose deposits in the leaves. Treatment with volatiles increased callose deposition in the leaves by up to five-fold, which promoted a rapid defense reaction that inhibited the incidence of gray mold by reinforcing cell wall. Taken together, our results show that B. methylotrophicus M4-96 promotes growth and induces systemic resistance in strawberry plants.

     

Biocontrol of Ascochyta blight by Azospirillum sp. depending on the degree of resistance of chickpea genotypes

Throughout arable land that was devoted to chickpea (Cicer arietinum L. (Family: Leguminosae) production, Ascochyta blight (Ascochyta rabiei (Pass.) L. (Order: Sphaeriales; Family: Mycosphaerellaceae) is a widespread disease that would lead to significant loss of chickpea yield. This study's purpose was to explain the responses of a resistant chickpea cultivar (ICC 12004) and a susceptible cultivar (Bivanij) in terms of disease resistance, disease symptoms appearance and expression pattern of two defence‐related genes (DEF0442 and Snakin2) after the Azospirillum brasilense seeds inoculation. In this research, the Snakin2 gene expression was affected by Azospirillum inoculation. The gene expression has been enhanced in plants inoculated with Azospirillum in both cultivars in comparison with non‐inoculated plants, but this change in ICC 12004 and Bivanij were significant and non‐significant, respectively. Although, Azospirillum would up regulate the DEF0422 gene expression in ICC 12004, but it would down regulate the expression of this gene in Bivanij. A. brasilense inoculation decreased the A. rabiei disease severity, regardless of the chickpea cultivar. Bivanij still could be classified as susceptible, even if treated with A. brasilense.     

Impaired sterol ester synthesis alters the response of Arabidopsis thaliana to Phytophthora infestans

Non‐host resistance of Arabidopsis thaliana against Phytophthora infestans, the causal agent of late blight disease of potato, depends on efficient extracellular pre‐ and post‐invasive resistance responses. Pre‐invasive resistance against P. infestans requires the myrosinase PEN2. To identify additional genes involved in non‐host resistance to P. infestans, a genetic screen was performed by re‐mutagenesis of pen2 plants. Fourteen independent mutants were isolated that displayed an enhanced response to Phytophthora (erp) phenotype. Upon inoculation with P. infestans, two mutants, pen2‐1 erp1‐3 and pen2‐1 erp1‐4, showed an enhanced rate of mesophyll cell death and produced excessive callose deposits in the mesophyll cell layer. ERP1 encodes a phospholipid:sterol acyltransferase (PSAT1) that catalyzes the formation of sterol esters. Consistent with this, the tested T‐DNA insertion lines of PSAT1 are phenocopies of erp1 plants. Sterol ester levels are highly reduced in all erp1/psat1 mutants, whereas sterol glycoside levels are increased twofold. Excessive callose deposition occurred independently of PMR4/GSL5 activity, a known pathogen‐inducible callose synthase. A similar formation of aberrant callose deposits was triggered by the inoculation of erp1 psat1 plants with powdery mildew. These results suggest a role for sterol conjugates in cell non‐autonomous defense responses against invasive filamentous pathogens.     

Effect of lead (Pb) on the systemic movement of RNA viruses in tobacco (Nicotiana tabacum var. Turkish)

Effect of various lead (Pb) concentrations on the systemic movement of RNA viruses was examined in tobacco plants. Prior to inoculation, plants were grown hydroponically for 6 days in Hoagland’s solution supplemented with five concentrations of lead nitrate [Pb(NO₃)₂]: 0.0 (control), 10, 15, 50, and 100 μM. Four different RNA viruses with different cell-to-cell movement mechanisms were used. Two weeks after inoculation lower and upper leaves of each treatment were harvested and examined for the presence of viral coat protein. In plants inoculated with Tobacco mosaic virus, Potato virus X, and Tobacco etch virus, TEM images and western blot assays confirmed the presence of viral coat proteins in the upper leaves of all lead treatments. However, in plants inoculated with Turnip vein-clearing virus (TVCV), no signs of viral particles were detected in the upper leaves of plants treated with 10 μM or 15 μM lead nitrate. In contrast, plants treated with high concentrations of lead nitrate (50 μM or 100 μM) showed viral particles in their upper leaves. In plants treated with 10 μM or 15 μM lead nitrate, callose accumulation was the same as in control plants. This suggests that non-toxic concentrations of lead nitrate may trigger the production of putative cellular factors in addition to callose that interfere with the TVCV systemic movement. In contrast, plants treated with 100 μM lead nitrate showed less callose as compared to control plants, facilitating the systemic movement of TVCV.     

Callose Synthase in Pinus sylvestris Response during Infection by Species of Heterobasidion annosum sensu lato with Varied Host Preferences

Strengthening of plant cell walls at the site of fungal entry is one of the earliest plant responses to fungal pathogens. The aim of our study was to characterize the pattern of callose synthase localization and callose deposition in roots of Pinus sylvestris after infection by species of the Heterobasidion annosum s.l. complex with different host specificity: H. annosum s.s., H. parviporum and H. abietinum. To address this, sense‐labelled probes and ribonuclease‐treated samples were used to determine in situ hybridizations of callose synthase by FISH method. Furthermore, determination of callose accumulation within P. sylvestris cells was carried out using aniline blue. The different species of H. annosum s.l. had distinct impacts on the callose synthase staining within plant tissues. Moreover, while inoculation with strains of H. abietinum resulted in callose synthase accumulation at the point of hyphae contact with the host cell, this was not observed with the other species. A significant difference in callose synthesis localization was observed after inoculation with varied species of H. annosum s.l. as a result of the specific interactions with the host.     

Changes in nitric oxide levels and their relationship with callose deposition during the interaction between soybean and Soybean mosaic virus

• The present study aimed to investigate changes in nitric oxide (NO) level and its relationship with callose deposition during the interaction between soybean and Soybean mosaic virus (SMV).
• Soybean cv. ‘Jidou 7’ and SMV strains N3 and SC‐8 were used to constitute incompatible and compatible combinations. Intracellular NO was labelled with the NO‐specific fluorescence probe DAF‐FM DA. Confocal laser scanning microscopy (CLSM) was then used to observe changes in NO production during SMV infection‐induced defence responses in soybean.
• The results showed NO fluorescence increased rapidly at 2–72 h post‐inoculation, peaked at 72 h and then decreased in the incompatible combination. However, in the compatible combination, extremely weak NO fluorescence appeared in the early stage (2–24 h) post‐inoculation, but was not observed thereafter. Injections of the NO scavenger c‐PTIO prior to inoculation postponed the onset of NO production to 48 or 72 h post‐inoculation. The same occurred when injections of NR or NOS inhibitors were applied prior to inoculation. The observation of callose fluorescence in the incompatible combination revealed that either the elimination or reduction of NO in the early stage led to a delay in callose formation, enabling the virus to cause systemic infection.
• Together with our previous findings, this study indicates that viral infection could induce NO production and callose deposition during the incompatible interaction between soybean and SMV. The production of NO involves NR and NOS enzymatic pathways, and NO mediates the process of callose deposition at plasmodesmata.

     

Natural occurrence of Azospirillum brasilense in strawberry plants

Azospirillum species are free-living nitrogen-fixing bacteria commonly found in soil and in association with roots of different plant species. For their capacity to stimulate growth they are known as plant growth-promoting bacteria (PGPB). In this work, we demonstrate the natural occurrence and colonization of different parts of strawberry plants by Azospirillum brasilense in the cropping area of Tucumán, Argentina. Although bacteria isolations were carried out from two strawberry cultivars, e.g., Camarosa and Pájaro, attempts were successful only with the cultivar Camarosa. Whereas different strains of Azospirillum were isolated from the root surface and inner tissues of roots and stolons of the cultivar Camarosa, we have not obtained Azospirillum isolates from the cultivar Pájaro. After microbiological and molecular characterization (ARDRA) we determined that the isolates belonged to the species A. brasilense. All isolates showed to have the capacity to fix nitrogen, to produce siderophores and indoles. Local isolates exhibited different yields of indoles production when growing in N-free NFb semisolid media supplemented or not with tryptophan (0.1 mg ml⁻¹). This is the first report on the natural occurrence of A. brasilense in strawberry plants, especially colonizing inner tissues of stolons, as well as roots. The local isolates showed three important characteristics within the PGPB group: N₂-fixation, siderophores, and indoles production.     

Azospirillum brasilense ameliorates the response of Arabidopsis thaliana to drought mainly via enhancement of ABA levels

Production of phytohormones is one of the main mechanisms to explain the beneficial effects of plant growth‐promoting rhizobacteria (PGPR) such as Azospirillum sp. The PGPRs induce plant growth and development, and reduce stress susceptibility. However, little is known regarding the stress‐related phytohormone abscisic acid (ABA) produced by bacteria. We investigated the effects of Azospirillum brasilense Sp 245 strain on Arabidopsis thaliana Col‐0 and aba2‐1 mutant plants, evaluating the morphophysiological and biochemical responses when watered and in drought. We used an in vitro‐grown system to study changes in the root volume and architecture after inoculation with Azospirillum in Arabidopsis wild‐type Col‐0 and on the mutant aba2‐1, during early growth. To examine Arabidopsis development and reproductive success as affected by the bacteria, ABA and drought, a pot experiment using Arabidopsis Col‐0 plants was also carried out. Azospirillum brasilense augmented plant biomass, altered root architecture by increasing lateral roots number, stimulated photosynthetic and photoprotective pigments and retarded water loss in correlation with incremented ABA levels. As well, inoculation improved plants seed yield, plants survival, proline levels and relative leaf water content; it also decreased stomatal conductance, malondialdehyde and relative soil water content in plants submitted to drought. Arabidopsis inoculation with A. brasilense improved plants performance, especially in drought.     

Functional analysis of endo‐1,4‐β‐glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant–pathogen interactions

Plant cell wall modification is a critical component in stress responses. Endo‐1,4‐β‐glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence‐signalling network. A study of a set of Arabidopsis EG T‐DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant–pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant–pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.     

The impact of Pseudomonas syringae type III effectors on transient protein expression in tobacco

The production of recombinant proteins in plants is often achieved by transient expression, e.g. following the injection or vacuum infiltration of Agrobacterium tumefaciens into tobacco leaves. We investigated the associated plant defence responses, revealing that callose deposition is triggered by T–DNA transfer and that subsets of secondary metabolites accumulate in response to mechanical wounding or the presence of bacteria. We also tested the ability of five co‐expressed type III effector proteins from Pseudomonas syringae to modulate these defence responses and increase the yield of two model proteins, the fluorescent marker DsRed and monoclonal antibody 2G12. HopF2 and AvrRpt2 induced necrotic lesions 5 days post‐injection (dpi) even at low doses (OD_600 nm = 0.0078), and increased the concentration of certain secondary metabolites. HopAO1 significantly reduced the number of callose deposits at 2 dpi compared to cells expressing DsRed and 2G12 alone, whereas HopI1 reduced the concentration of several secondary metabolites at 5 dpi compared to cells expressing DsRed and 2G12 alone. Co‐expression with HopAO1, AvrPtoB or HopI1 increased the concentrations of DsRed and 2G12 increased by ~6% but this was not a significant change. In contrast, HopF2 and AvrRpt2 significantly reduced the concentrations of DsRed and 2G12 by 34% and 22%, respectively. Our results show that type III effector proteins can modulate plant defence responses and secondary metabolite profiles but that transient co‐expression is not sufficient to increase the yields of target recombinant proteins in tobacco.     

Arbuscular mycorrhiza‐mediated resistance in tomato against Cladosporium fulvum‐induced mould disease

Root colonization with arbuscular mycorrhizal fungi (AMF) enhances plant resistance particularly against soil‐borne pathogenic fungi. In this study, mycorrhizal inoculation with Glomus mosseae (Gm) significantly alleviated tomato mould disease caused by the air‐borne fungal pathogen, Cladosporium fulvum (Cf). The disease index (DI) in local leaves (receiving pathogen inoculation) and systemic leaves (just above the local leaf without pathogen inoculation) was 36.4% and 11.7% in mycorrhizal plants, respectively, whereas DI was 59.6% and 36.4% in the corresponding leaves of AMF non‐inoculated plants, after 50 days of Gm inoculation, corresponding to 15 days after Cf inoculation by leaf infiltration. Foliar spray inoculation with Cf also revealed that AMF pre‐inoculated plants had a higher resistance against subsequent pathogen infection, where the DI was 41.3% in mycorrhizal plants vs. 64.4% in AMF non‐inoculated plants. AMF‐inoculated plants showed significantly higher fresh and dry weight than non‐inoculated plants under both control (without pathogen) and pathogen treatments. AMF‐inoculated plants exhibited significant increases in activities of superoxide dismutase and peroxidase, along with decreases in levels of H₂O₂ and malondialdehyde, compared with non‐inoculated plants after pathogen inoculation. AMF inoculation led to increases in total chlorophyll contents and net photosynthesis rate as compared with non‐inoculated plants under control and pathogen infection. Pathogen infection on AMF non‐inoculated plants led to decreases in chlorophyll fluorescence parameters. However, pathogen infection did not affect these parameters in mycorrhizal plants. Taken together, these results indicate that AMF colonization may play an important role in plant resistance against air‐borne pathogen infection by maintaining redox poise and photosynthetic activity.     

INVOLVEMENT OF INDOLE‐3‐ACETIC ACID PRODUCED BY THE GROWTH‐PROMOTING BACTERIUM AZOSPIRILLUM SPP. IN PROMOTING GROWTH OF CHLORELLA VULGARIS1

Involvement of indole‐3‐acetic acid (IAA), produced by the microalgae‐growth‐promoting bacteria Azospirillum brasilens and A. lipoferum, in promoting growth of the microalga Chlorella vulgaris Beij. was studied. Four wildtype strains of Azospirillum and their IAA‐deficient mutants were co‐immobilized with C. vulgaris in alginate beads. Cultures were grown in synthetic growth medium supplemented with tryptophan. Growth promotion of microalgae and production of exogenous IAA by Azospirillum spp. were monitored. All wildtype Azospirillum spp. produced significant but varying amounts of IAA, while their mutant forms produced significantly less. The results demonstrated a significant growth promotion in Chlorella cultures when immobilized with the four wildtype strains of Azospirillum, while very low or no enhanced growth was induced by the four IAA‐deficient mutants, compared to when C. vulgaris is immobilized alone. A complementation experiment, where an IAA‐attenuated mutant (A. brasilense SpM7918) was supplemented with IAA produced by its parental wildtype strain (A. brasilense Sp6), restored growth promotion in the microalgae‐mutant culture.     

Nitrogen fixation associated with grasses and cereals: Recent results and perspectives for future research

Over the last few years research in the area of biological nitrogen fixation (BNF) associated with cereals and grasses has become divided into two areas. On the one hand there have been a large number of reports of responses of field-grown plants to inoculation with N₂-fixing bacteria, principallyAzospirillum spp. On the other hand there have been several reports of significant contributions of associated BNF to the nutrition of several crops, including wetland rice, sugar cane and some forage grasses. However, where BNF contributions have definitely been established no certain information is available as to the diazotrophic organisms responsible. Furthermore, certain recent reports indicate that, at least in some cases, responses of plants to inoculation withAzospirillum spp. have been shown not to be due to BNF contributions. In this paper we review some recent progress in this field, particularly at our institute in Rio de Janeiro, concerning specificity of selected Azospirillum strains in the infection of cereal roots and the promotion of responses in the host plants. The possible mechanisms of plant response are discussed including the possibility that plant growth substances or bacterial nitrate reductase are involved. The application of¹⁵N and N balance techniques to the quantification of plant associated BNF are considered and the possible strategies that may be adopted to further the understanding of true N₂-fixing plant/diazotroph associations. The recent discovery of many more plant-associated N₂-fixing bacteria suggests that further research in this area may eventually lead to the development of such associations with applications for agricultural productivity.     

A novel Meloidogyne graminicola effector,MgMO237, interacts with multiple host defence‐related proteins to manipulate plant basal immunity and promote parasitism

Plant‐parasitic nematodes can secrete effector proteins into the host tissue to facilitate their parasitism. In this study, we report a novel effector protein, MgMO237, from Meloidogyne graminicola, which is exclusively expressed within the dorsal oesophageal gland cell and markedly up‐regulated in parasitic third‐/fourth‐stage juveniles of M. graminicola. Transient expression of MgMO237 in protoplasts from rice roots showed that MgMO237 was localized in the cytoplasm and nucleus of the host cells. Rice plants overexpressing MgMO237 showed an increased susceptibility to M. graminicola. In contrast, rice plants expressing RNA interference vectors targeting MgMO237 showed an increased resistance to M. graminicola. In addition, yeast two‐hybrid and co‐immunoprecipitation assays showed that MgMO237 interacted specifically with three rice endogenous proteins, i.e. 1,3‐β‐glucan synthase component (OsGSC), cysteine‐rich repeat secretory protein 55 (OsCRRSP55) and pathogenesis‐related BetvI family protein (OsBetvI), which are all related to host defences. Moreover, MgMO237 can suppress host defence responses, including the expression of host defence‐related genes, cell wall callose deposition and the burst of reactive oxygen species. These results demonstrate that the effector MgMO237 probably promotes the parasitism of M. graminicola by interacting with multiple host defence‐related proteins and suppressing plant basal immunity in the later parasitic stages of nematodes.     

Histochemical Studies of the Non‐Host Resistance and the Role of Actin Cytoskeleton in Pepper Against Colletotrichum orbiculare

The penetration process and defence reactions (hypersensitive response, oxidative burst and cell wall fortification) of Colletotrichum orbiculare were studied histochemically on pepper cultivar ‘A11’ (non‐host) and susceptible cucumber cultivar ‘Changchun Thorn’ (host). The results indicate that C. orbiculare could hardly penetrate the non‐host pepper leaves. It was papillae rather than hypersensitive response and H₂O₂ that played an important role in resisting the colonization and development of C. orbiculare on the non‐host pepper. The depolymerization of the actin microfilament weakened the papilla deposition of pepper and allowed successful penetration of the non‐adapted C. orbiculare, suggesting that the actin cytoskeleton of pepper is significant in preventing the invasion of the non‐host pathogen C. orbiculare.     

Cell wall biochemical alterations during Agrobacterium‐mediated expression of haemagglutinin‐based influenza virus‐like vaccine particles in tobacco

Influenza virus‐like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant‐based biotechnology allows for the large‐scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium‐mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post‐Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG‐I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin‐based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing.