A fungal protein elicitor PevD1 induces Verticillium wilt resistance in cotton

Key message

We found that the elicitor PevD1 triggered innate immunity in cotton, which plays an important role in future cotton wilt disease control.

Abstract

Elicitors can induce defense responses in plants and improve pathogen resistance. PevD1 is a secreted protein from Verticillium dahliae and activates the hypersensitive response and systemic acquired resistance to tobacco mosaic virus in tobacco plants. To investigate the PevD1-induced disease resistance mechanisms in cotton (Gossypium hirsutum), we report that Escherichia coli expressing PevD1 enhanced cotton resistance and the defense response to the fungal pathogen V. dahliae. The results showed that recombinant PevD1 improved cotton resistance when infiltrated at a concentration as low as 4 μg ml^?1, and the highest disease reduction was 38.16 % on the 15th day post V. dahliae inoculation. This protein was able to systemically induce hydrogen peroxide production, nitric oxide generation, lignin deposition, vessel reinforcement and defense enzymes, including phenylalanine ammonia-lyase, peroxidase, and polyphenol oxidase. PevD1 also enhanced the expression of three pathogenesis-related genes, namely, β-1,3-glucanase, chitinase, and cadinene synthase, and three key genes, PAL, C4H1, and 4CL, from the cotton defense phenylpropanoid metabolism pathway. Our results demonstrated that PevD1 acted as an effector in cotton and V. dahliae interactions and triggered innate immunity in cotton, resulting in the upregulation of defense-related genes, metabolic substance deposition and cell wall modifications. PevD1 is a candidate plant defense activator for cotton wilt disease control.     

Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection

Key message

We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics.

Abstract

Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12–16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1–2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying.     

Comparative root colonisation of strawberry cultivars Camarosa and Festival by Fusarium oxysporum f. sp. fragariae

Background and aims

Strawberry (Fragaria x ananassa) is a high-value crop worldwide. Fusarium oxysporum f. sp. fragariae causes rapid wilting and death of strawberry plants and severe economic losses worldwide. To date, no studies have been conducted to determine colonisation of either susceptible or resistant strawberry plants by F. oxysporum f. sp. fragariae, or whether plant colonisation by F. oxysporum f. sp. fragariae differs between susceptible and resistant cultivars.

Methods

Colonisation of strawberry plants by a pathogenic isolate of F. oxysporum f. sp. fragariae was examined both on the root surface and within root tissue of one resistant cv. Festival and one susceptible cv. Camarosa using light and scanning electron microscopy from 4?h to 7?d post inoculation (pi).

Results

Resistant cv. Festival significantly impeded the spore germination and penetration from 4 to 12 hpi and subsequent growth and colonisation by this pathogen until 7 dpi compared with susceptible cv. Camarosa. At 7 dpi, fungal colonisation in resistant cv. Festival remained mainly confined to the epidermal layer of the root, while in susceptible cv. Camarosa, hyphae not only had heavily colonised the cortical tissue throughout but had also colonised vascular tissues.

Conclusions

This study demonstrates for the first time that resistance of a strawberry cultivar to F. oxysporum f. sp. fragariae is a result of impedance of pathogen growth and colonisation both on the plant surface and within host tissues. Resistance mechanisms identified in this study will be of high value for breeding programmes in developing new disease-resistant cultivars to manage this serious strawberry disorder.     

A wood based low-temperature biochar captures NH3-N generated from ruminant urine-N, retaining its bioavailability

Aims

Ammonia (NH₃) can be volatilised from the soil surface following the surface application of nitrogenous fertilisers or ruminant urine deposition. The volatilisation of NH₃ is of agronomic and environmental concern, since NH₃-N is a form of reactive nitrogen. Ammonia adsorption onto biochar has the potential to mitigate NH₃ losses, but to date no studies have examined the potential for reducing NH₃ losses when biochar is present in the soil matrix.

Methods

We used ¹⁵N-enriched urine to examine the effect of incorporating a wood based low-temperature biochar into soil on NH₃ volatilisation. Then, we extracted the urine-treated biochar and compared its potential to act as a plant N source with fresh biochar, while growing ryegrass (Lolium perenne).

Results

The NH₃ volatilisation from ¹⁵N-labelled ruminant urine, applied to soil, was reduced by 45% after incorporating either 15 or 30?t ha^?1 of biochar. When the urine-treated biochar particles were transferred into fresh soil, subsequent plant growth was not affected but the uptake of ¹⁵N in plant tissues increased, indicating that the adsorbed-N was plant available.

Conclusions

Our results show that incorporating biochar into the soil can significantly decrease NH₃ volatilisation from ruminant urine and that the NH₃-N adsorbed onto the biochar is bioavailable. Further studies are now required to assess the temporal dynamics of the N pools involved.     

Arabidopsis Brassinosteroid-overproducing gulliver3-D/dwarf4-D mutants exhibit altered responses to Jasmonic acid and pathogen

Key message

Arabidopsis gulliver3 - D/dwarf4 - D displays growth-promoting phenotypes due to activation tagging of a key brassinosteroid biosynthetic gene DWARF4. In gul3-D/dwf4-D , the Jasmonate and Salicylate signaling pathways were relatively activated and suppressed, respectively.

Abstract

Energy allocation between growth and defense is elegantly balanced to achieve optimal development in plants. Brassinosteroids (BRs), steroidal hormones essential for plant growth, are regulated by other plant hormones, including auxin and jasmonates (JA); auxin stimulates the expression of a key brassinosteroid (BR) biosynthetic gene, DWARF4 (DWF4), whereas JA represses it. To better understand the interaction mechanisms between growth and defense, we isolated a fast-growing mutant, gulliver3-D (gul3-D), that resulted from the activation tagging of DWF4, and examined the response of this mutant to defense signals, including JA, Pseudomonas syringae pv. tomato (Pst DC3000) infection, and wounding. The degree of root growth inhibition following MeJA treatment was significantly decreased in gul3-1D/dwf4-5D relative to the wild type, suggesting that JA signaling is partially desensitized in gul3-1D. Quantitative RT-PCR analysis of the genes involved in JA and salicylic acid (SA) responses, including MYC2, PDF1.2, CORI3, PR1, and PR2, revealed that JA signaling was preferentially activated in gul3-1D, whereas SA signaling was suppressed. As a result, gul3-1D was more susceptible to a biotrophic pathogen, Pst DC3000. Based on our results, we propose a model in which BR and JA cooperate to balance energy allocation between growth and defense responses. In ambient conditions, BRs promote plant growth; however, when stresses trigger JA signaling, JA compromises BR signaling by downregulating DWF4 expression.     

Ectopic expression of a novel Ser/Thr protein kinase from cotton (Gossypium barbadense), enhances resistance to Verticillium dahliae infection and oxidative stress in Arabidopsis

Key message

Overexpression of a cotton defense-related gene GbSTK in Arabidopsis resulted in enhancing pathogen infection and oxidative stress by activating multiple defense-signaling pathways.

Abstract

Serine/threonine protein kinase (STK) plays an important role in the plant stress-signaling transduction pathway via phosphorylation. Most studies about STK genes have been conducted with model species. However, their molecular and biochemical characterizations have not been thoroughly investigated in cotton. Here, we focused on one such member, GbSTK. RT-PCR indicated that it is induced not only by Verticillium dahliae Kleb., but also by signaling molecules. Subcellular localization showed that GbSTK is present in the cell membrane, cytoplasm, and nucleus. Overexpression of GbSTK in Arabidopsis resulted into the enhanced resistance to V. dahliae. Moreover, Overexpression of GbSTK elevated the expression of PR4, PR5, and EREBP, conferring on transgenic plants enhanced reactive oxygen species scavenging capacity and oxidative stress tolerance. Our results suggest that GbSTK is active in multiple defense-signaling pathways, including those involved in responses to pathogen infection and oxidative stress.     

Endogenous jasmonic and salicylic acids levels in the Cd-hyperaccumulator Noccaea (Thlaspi) praecox exposed to fungal infection and/or mechanical stress

Key message

Sensitivity to Erysiphe in Noccaea praecox with low metal supply is related to the failure in enhancing SA. Cadmium protects against fungal-infection by direct toxicity and/or enhanced fungal-induced JA signaling.

Abstract

Metal-based defense against biotic stress is an attractive hypothesis on evolutionary advantages of plant metal hyperaccumulation. Metals may compensate for a defect in biotic stress signaling in hyperaccumulators (metal-therapy) by either or both direct toxicity to pathogens and by metal-induced alternative signaling pathways. Jasmonic acid (JA) and salicylic acid (SA) are well-established components of stress signaling pathways. However, few studies evaluate the influence of metals on endogenous concentrations of these defense-related hormones. Even less data are available for metal hyperaccumulators. To further test the metal-therapy hypothesis we analyzed endogenous SA and JA concentrations in Noccaea praecox, a cadmium (Cd) hyperaccumulator. Plants treated or not with Cd, were exposed to mechanical wounding, expected to enhance JA signaling, and/or to infection by biotrophic fungus Erysiphe cruciferarum for triggering SA. JA and SA were analyzed in leaf extracts using LC–ESI(?)–MS/MS. Plants without Cd were more susceptible to fungal attack than plants receiving Cd. Cadmium alone tended to increase leaf SA but not JA. Either or both fungal attack and mechanical wounding decreased SA levels and enhanced JA in the Cd-rich leaves of plants exposed to Cd. High leaf Cd in N. praecox seems to hamper biotic-stress-induced SA, while triggering JA signaling in response to fungal attack and wounding. To the best of our knowledge, this is the first report on the endogenous JA and SA levels in a Cd-hyperaccumulator exposed to different biotic and abiotic stresses. Our results support the view of a defect in SA stress signaling in Cd hyperaccumulating N. praecox.     

Biochar from Miscanthus: a potential silicon fertilizer

Background and aims

Silicon (Si) is largely recognized to improve plant growth subjected to various biotic and abiotic stresses. As plants accumulate Si in the form of readily-soluble phytolith, we examine the possibility of using phytolith-rich biochar as a bio-available Si source for increasing the agronomical productivity of Si high-accumulator plants while augmenting soil fertility and C sequestration.

Methods

By adding three different biochars (Miscanthus x giganteus straws, coffee husks and woody material) at two different concentrations (1 % and 3 %; w/w) to soil samples, we investigated the effects on the soil respiration, the chemical characteristics and the kinetic release of bio-available Si (CaCl₂-extractable Si).

Results

Here we show that the biochar from Miscanthus straws was the most attractive amendment. Its incorporation at a 3 % rate improved the soil fertility parameters (pH and available cations) and combined the highest mean residence time of carbon (C) in soil (MRT?=?50 years) with the highest rate of release of bio-available Si. We attribute this result to the presence of phytoliths in this biochar, as revealed by SEM-EDS analysis.

Conclusions

Not only did the biochar from Miscanthus enhance both soil C sequestration and fertility, but the results of this study suggest that it can also be considered as a potential source of bio-available Si. Although our conclusions should be substantiated in the field, we suggest that Miscanthus biochar could be used as a potential source of bio-available silicon for the culture of such crop as Si-accumulator plants growing, for instance, in highly weathered tropical soils with low content in carbon, nutrients and bio-available Si.     

Alleviation of salt stress of symbiotic Galega officinalis L. (goat's rue) by co-inoculation of Rhizobium with root-colonizing Pseudomonas

Background and aims

Salt stress is an increasing problem in agricultural soils in many parts of the world, and salt tolerant cropping systems are in great demand. We investigated the effect of co-inoculation of Galega officinalis with Rhizobium galegae and two plant growth promoting Pseudomonas species on plant growth, nodulation, and N content under salt stress.

Methods

The effect of inoculation with R. galegae sv. officinalis HAMBI 1141 alone and in combination with the root-colonizing Pseudomonas extremorientalis TSAU20 or Pseudomonas trivialis 3Re27 on the growth of G. officinalis exposed to salt stress (50 and 75 mM NaCl) was studied under gnotobiotic and greenhouse conditions.

Results

The growth of goat’s rue was reduced at 50 and 75 mM NaCl both in the gnotobiotic sand system and in low-fertilized potting soil in the greenhouse. Co-inoculation of unstressed and salt-stressed goat’s rue with R. galegae HAMBI 1141 and either P. extremorientalis TSAU20 or P. trivialis 3Re27 significantly improved root and shoot growth and increased nodulation of plant roots in both growth systems compared with plants inoculated with R. galegae alone. The nitrogen content of co-inoculated plant roots was significantly increased at 75 mM NaCl in potting soil. Co-inoculation of G. officinalis with either of the two plant growth promoting (PGPR) Pseudomonas strains also improved root tip-colonization by R. galegae cells.

Conclusions

The co-inoculation of goat’s rue with Rhizobium and PGPR Pseudomonas strains alleviated salt stress of plants grown in NaCl-amended gnotobiotic sand systems and in potting soil in the greenhouse.     

The 4-phosphopantetheinyl transferase of Trichoderma virens plays a role in plant protection against Botrytis cinerea through volatile organic compound emission

Aims

This work was conducted to examine the effects of volatile organic compounds (VOCs) from Trichoderma virens and the 4-phosphopantetheinyl transferase 1 (TvPPT1) mutant in growth promotion and induction of defense responses of Arabidopsis thaliana seedlings using a co-cultivation system in vitro.

Methods

The contribution of VOCs to plant development and immunity was assessed by comparing the effectiveness of WT and Δppt1 mutant strains of T. virens in the formation of lateral roots and protection conferred against Botrytis cinerea. VOCs released by T. virens and Δppt1 mutant were compared by gas chromatography–mass spectrometry.

Results

Plants exposed to volatiles from WT T. virens showed 2-fold increase in fresh weight when compared to axenically-grown seedlings, which correlated with increased root branching and enhanced expression of the jasmonic acid-responsive marker pLox2:uidA as well as accumulation of jasmonic acid and hydrogen peroxide. T. virens produced a series of hydrocarbon terpenes, including the sesquiterpenes β-caryophyllene, (?)-β-elemene, germacrene D, τ-cadinene, δ-cadinene, α-amorphene, and τ-selinene and the monoterpenes β-myrcene, trans-β-ocimene, and cis-β-ocimene that were absent in TvPPT1 mutant.

Conclusions

Our results indicate that T. virens VOCs elicit both development and defense programs and that PPT1 plays an important role in biosynthesis of terpenes and plant protection against B. cinerea.     

Phytohormones in plant root-Piriformospora indica mutualism

Piriformospora indica is a mutualistic root-colonising basidiomycete that tranfers various benefits to colonized host plants including growth promotion, yield increases as well as abiotic and biotic stress tolerance. The fungus is characterized by a broad host spectrum encompassing various monocots and dicots.^1,2 Our recent microarray-based studies indicate a general plant defense suppression by P. indica and significant changes in the GA biosynthesis pathway.³ Furthermore, barley plants impaired in GA synthesis and perception showed a significant reduction in mutualistic colonization, which was associated with an elevated expression of defense-related genes. Here, we discuss the importance of plant hormones for compatibility in plant root-P. indica associations. Our data might provide a first explanation for the colonization success of the fungus in a wide range of higher plants.Key words: compatibility, plant defense, gibberellic acid, symbiosis, plant hormones     

The Saharan isolate Saccharothrix algeriensis NRRL B-24137 induces systemic resistance in Arabidopsis thaliana seedlings against Botrytis cinerea

Background and aim

Saccharothrix algeriensis NRRL B-24137, isolated from a Saharan soil, has been described as a potential biocontrol agent against Botrytis cinerea and other phytopathogens. However, the plant protection mechanisms involved still need to be described. The aim of this study was to determine this protection phenomenon as well as parts of the mechanisms involved, using Arabidopsis thaliana seedlings and B. cinerea.

Methods

The bacterial colonization process was evaluated on A. thaliana seedlings using fluorescence in situ hybridization. Protection of A. thaliana seedlings inoculated with NRRL B-24137 against B. cinerea was then evaluated. Parts of the mechanisms involved in the systemic protection against B. cinerea were evaluated using known mutants of genes involved in jasmonate (JA)/ethylene (ET)/salicylic acid (SA) signaling. Other Arabidopsis mutants, AtrhbohD-3, AtrhbohF-3, and ups1-1 were also screened to determine other parts of the mechanisms involved.

Results

The results showed that the strain NRRL B-24137 colonized, epi- and endophytically, the roots of Arabidopsis seedlings but the strain was not a systemic colonizer during the time of the experiment. The strain NRRL B-24137 also reduced B. cinerea symptoms and the protection was linked to known mechanisms of induced systemic resistance (ISR; JA/ET signaling), as well as to functionality of AtrbohF oxidase and of UPS1. Crosstalk between ET/JA and SA signaling could also be involved.

Conclusions

The isolate NRRL B-24137, after colonizing the root systems of A. thaliana, induces an ISR against B. cinerea, which is JA/ET dependent, but could also require SA crosstalk and protection could also require NAPDH oxidases and UPS1 functionalities.