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Quantification of necrotic activity by electrolyte leakage assay induced by DserNEP proteins in micropropagated plants (A), and viability of V. vinifera cell cultures treated with DserNEP proteins determined 5 days after protein exposition by staining with FDA (B). DserNEP1 was assayed at 0.05 and 0.1 mg/mL, DserNEP2 at 0.25 and 0.5 mg/mL, and control leaves were inoculated with water. Data shown represent the mean ± SD from three independent experiments. Bars marked with the same letter do not differ at P = 0.05.
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Many phytopathogenic fungi produce necrosis and ethylene inducing peptide 1 (Nep1-like proteins or NLP) that trigger leaf necrosis and the activation of defense mechanisms. These proteins have been widely studied in plant pathogens as Moniliophthora perniciosa or Botrytis cinerea between others, but little is known about their biological roles in g...
Citations
... Effector genes are also known as avirulence genes (Avr), while host plants possess resistance genes (R) . The interaction between plantpathogenic fungi and host plants can be understood as the interaction between Avr and R genes (Cobos et al. 2019). Recognition of Avr gene products by R gene products leads to incompatibility and disease resistance, while nonrecognition results in compatibility and disease (Yu et al. 2019). ...
Integrated pest and disease management (IPDM) is a strategic approach that combines multiple pest and pathogen control methods to optimize their reduction while minimizing ecological and economic consequences. This multifaceted strategy serves as a fundamental component of sustainable agricultural systems, emphasizing the balanced integration of various methods to achieve effective and environmentally responsible pest and pathogen suppression. Modern agricultural practices, characterized by intensified production and monoculture systems, create optimal environments for pathogen proliferation and virulence. These conditions necessitate the IPDM strategies. Integrated pest and disease management is crucial for mitigating pathogen-induced losses and ensuring sustainable agricultural production. It aims to minimize reliance on chemical fungicides by promoting environment-friendly and economically viable strategies for disease control. This review delves into the major pathogens that affect the plants and the intricate relationship between IPDM and sustainable agriculture, examining the key principles, strategies, and benefits associated with integrating these disease management practices into the agricultural system. It underscores the crucial role of IPDM in minimizing environmental impacts, protecting beneficial organisms, fostering genetic diversity, and ensuring economic sustainability. By adopting integrated pest and disease management strategies, farmers can effectively manage plant diseases while simultaneously safeguarding the long-term health and productivity of their agricultural systems.
... For example, the removal of NLP-encoding genes did not change the virulence of Magnaporthe oryzae [7] and Botrytis elliptica [8] mutants. Through functional analyses, some members of the prolific NLP superfamilies of both Diplodia seriata [9] and Neofusicoccum parvum [10] were shown to exhibit varying levels of cytotoxicity. Contrary to their cytolytic counterpart, the noncytolytic NLPs cannot permeabilize the plant membrane but retain the capability of triggering plant immune responses; the biological role of the noncytolytic NLPs is yet to be characterized [11][12][13][14]. ...
... These findings agree with a recent study that describe some plant pathogens (necrotrophs and hemibiotrophs) detaining more NLP copies and a broad distribution of NLPs across saprotrophic species [3]. Cell death-inducing proteins, such as NLPs, which act at the plant apoplast level, are essential for host colonization [9,10,36] and may contribute to the decay of plant material [3]. Moreover, the role of the larger NLP family in necrotrophic plant pathogens, such as that of the Botryosphaeriales, can go beyond differential cytotoxicity and detain levels of functional diversification at different life stages [37,38] or, to some extent, contribute to infection of wider host ranges by those pathogens [37,39]. ...
... The only NLP from Zymoseptoria tritici (Dothideomycetes), which induced defense responses and cell death in dicots but not in monocots [42] and shared homology to members of the NLP1.1 subfamily. Paralogs of the NLP1.1 subfamily of Diplodia seriata (Dothideomycetes) encoded proteins that showed distinct levels of cytotoxicity to grapevine leaves [9]. ...
Necrosis and Ethylene-inducing peptide 1-like proteins (NLPs) are broadly distributed across bacteria, fungi, and oomycetes. Cytotoxic NLPs are usually secreted into the host apoplast where they can induce cell death and trigger plant immune responses in eudicots. To investigate the evolutionary history of the NLPs, we accessed the genomic resources of 79 species from 15 orders of Dothideomycetes. Phylogenetic approaches searched for biased patterns of NLP gene evolution and aimed to provide a phylogenetic framework for the cytotoxic activities of NLPs. Among Dothideomycetes, the NLP superfamily sizes varied, but usually contained from one to six members. Superfamily sizes were higher among pathogenic fungi, with family members that were mostly putative-effector NLPs. Across species, members of the NLP1 family (Type I NLPs) were predominant (84%) over members of the NLP2 family (Type II NLPs). The NLP1 family split into two subfamilies (NLP1.1 and NLP1.2). The NLP1.1 subfamily was broadly distributed across Dothideomycetes. There was strong agreement between the phylogenomics of Dothideomycetes and the phylogenetic tree based on members of the NLP1 subfamilies. To a lesser extent, phylogenomics also agreed with the phylogeny based on members of the NLP2 family. While gene losses seem to have shaped the evolutionary history of NLP2 family, ancient gene duplications followed by descent with modification characterized the NLP1 family. The strongest cytotoxic activities were recorded on NLPs of the NLP1.1 subfamily, suggesting that biased NLP gene retention in this subfamily favored the cytotoxic paralogs.
... To comparatively evaluate the necrosis-inducing activity of NLPs, five CaNLPs belonging to different types were prepared as previously described (Cobos et al., 2019). CaNLP4 represents the NLP with substitution of residues known to be essential for necrosis induction activity. ...
... The Agrobacterium-mediated transient gene expression method is widely used in the literature, but this method is mainly applied to model plants, such as Nicotiana species (Chen et al., 2021;Duhan et al., 2021). The infiltration of purified proteins method has also been used for the activity evaluation of NLPs in some forest plants (Cobos et al., 2019;Hunziker et al., 2021;Liu et al., 2021). In the present study, we employed purified recombinant protein infiltration into the leaf apoplast to analyze cytotoxic activity of NLPs, and obtained consistent results after repeated experiments. ...
Colletotrichum australisinense , a member of the Colletotrichum acutatum species complex, is an important pathogen causing rubber tree anthracnose. Genome-wide comparative analysis showed this species complex contains more genes encoding necrosis- and ethylene-inducing peptide 1-like proteins (NLPs) than other Colletotrichum species complexes, but little is known about their necrosis-inducing roles in host. The aim of this study was to analyze NLPs number and type in C. australisinense , and characterize their necrosis-inducing activity in host or non-host. According to phylogenetic relationship, conserved the cysteine residues and the heptapeptide motif (GHRHDWE), 11 NLPs were identified and classified into three types. Five of the eleven NLPs were evaluated for necrosis-inducing activity. CaNLP4 (type 1) could not induce necrosis in host or non-host plants. By contrast, both CaNLP5 and CaNLP9 (type 1) induced necrosis in host and non-host plants, and necrosis-inducing activity was strongest for CaNLP9. CaNLP10 (type 2) and CaNLP11 (type 3) induced necrosis in host but not non-host plants. Substitution of key amino acid residues essential for necrosis induction activity led to loss of CaNLP4 activity. Structural characterization of CaNLP5 and CaNLP9 may explain differences in necrosis-inducing activity. We evaluated the expression of genes coding CaNLP by reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) at different time-points after pathogen infection. It was found that genes encoding CaNLPs with different activities exhibited significantly different expression patterns. The results demonstrate that CaNLPs are functionally and spatially distinct, and may play different but important roles in C. australisinense pathogenesis.
... Botryosphaeriaceae species are known to cause foliar symptoms but, to date, they have never been isolated from leaves or fruits. Thus, it has been postulated that these symptoms are due to phytotoxins and secreted proteins (SP) produced by the fungi in the perennial part of the plant and then translocated to the leaves through the transpiration stream [31,32]. Moreover, for many pathogens, particularly in fungi and oomycetes, the excretion of proteins is essential for pathogenicity [33]. ...
... NLPs have two conserved domains in their amino acid sequence: two cysteines connected with a disulfide bond and a hepta-peptide (GHRHDWE), forming a negatively-charged cavity. These two elements are particularly essential for plant cell membrane damaging [32,[40][41][42]. It has been further demonstrated that these NLPs can cause cell death and ethylene production during the interaction between dicotyledonous and fungi [40][41][42]. ...
... Cobos and colleagues (2019) identified four NLP-like proteins in D. seriata. They demonstrated that these NLPs could play a role in the pathogenicity of D. seriata, inducing necrosis on the leaf margin that progressed through the center of the leaves of infected grapevine [32]. Interestingly, in the same study, the authors were able to note an improvement in the conductance of leaves where NLPs had been infiltrated. ...
Botryosphaeriaceae are a family of fungi associated with the decay of a large number of woody plants with economic importance and causing particularly great losses in viticulture due to grapevine trunk diseases. In recent years, major advances in the knowledge of the pathogenicity factors of these pathogens have been made possible by the development of next-generation sequencing. This review highlights the knowledge gained on genes encoding small secreted proteins such as effectors, carbohydrate-associated enzymes, transporters and genes associated with secondary metabolism, their representativeness within the Botryosphaeriaceae family and their expression during grapevine infection. These pathogenicity factors are particularly expressed during host–pathogen interactions, facilitating fungal development and nutrition, wood colonization, as well as manipulating defense pathways and inducing impacts at the cellular level and phytotoxicity. This work highlights the need for further research to continue the effort to elucidate the pathogenicity mechanisms of this family of fungi infecting grapevine in order to improve the development of control methods and varietal resistance and to reduce the development and the effects of the disease on grapevine harvest quality and yield.
... As an important marker in determining cell membrane integrity, electrolyte leakage due to the effect of RST has been previously reported in rice Sriram et al., 2000;Vidhyasekaran et al., 1997). Electrolyte leakage has also been recently used for necrosis quantification in grapevine infected by the necrotrophic pathogen Diplodia seriata (Cobos et al., 2019). ...
Aim:
To understand the mechanism of necrosis incited by a host-selective phytotoxin designated as Rhizoctonia solani toxin (RST) identified to be a potential pathogenic factor of Rhizoctonia solani AG1 IA, causing sheath blight (ShB) of rice.
Methods and results:
The metabolomic changes induced by the phytotoxic metabolite in a ShB susceptible rice cultivar were elucidated by Gas Chromatography-Mass Spectrometry (GC-MS) analysis and compared with that of the pathogen to identify rice metabolites targeted by the phytotoxin. The profiles of about 29 metabolites with various physiological roles in rice plants have been identified worldwide. Unsupervised and supervised multivariate chemometrics (Principal Component Analysis, PCA and Partial Least Squares-Discriminant Analysis, PLS-DA) and cluster (Heat maps) analyses were used to compare the metabolites obtained from chemical profiles of the treatments with sterile distilled water (SDW) control. The results indicated that the rice plant expressed more metabolites in response to the pathogen than the phytotoxin and was lowest in SDW control. The key metabolites expressed in rice in response to the treatments were investigated by the Variable Importance in Projection (VIP) analysis using P< 0.05 VIP >15. The analysis identified 7 and 11 upregulating metabolites in the phytotoxin and the pathogen treatments, respectively, compared to the untreated control. Among the phytotoxin-treated and the pathogen inoculated samples, the phytotoxin treated sample recorded upregulation of 6 metabolites, whereas 9 metabolites were upregulated in the pathogen inoculated samples. These upregulating metabolites are speculated for the necrotic symptoms characteristic to both the phytotoxin and pathogen. In this analysis, hexadecanoic acid and dotriacontane were highly expressed metabolites specific to the phytotoxin and pathogen-treated samples, respectively. Besides upregulation, the metabolites also have a VIP score of >1.5 and hence fulfilled the criteria of classifying them as reliable potential biomarkers. In the pathway analysis, hexadecanoic acid and dotriacontane were identified to be involved in several important biosynthetic pathways of rice, such as the biosynthesis of saturated fatty acid and unsaturated fatty acids cutin, suberin, and wax.
Conclusions:
The study concludes that though certain metabolites induced by the phytotoxin in the susceptible variety during necrosis shares with that of the pathogen, the identification of metabolites specific to the phytotoxin in comparison to the pathogenic and SDW controls indicated that the phytotoxin modulates the host metabolism differently and hence can be a potential pathogenicity factor of the ShB fungus.
Significance and impact of the study:
Due to lack of knowledge on the pathway genes of RST and in the absence of an ShB resistant variety, understanding differentially expressed metabolic changes induced in the susceptible variety by the phytotoxin in comparison to that of the pathogenic and uninoculated controls enables us to identify the key metabolite changes during the ShB infection. Such metabolomic changes can further be used to infer gene functions for exploitation in ShB control.
... NLPs are taxonomically widespread in fungi, oomycetes, and bacteria [49,50]. The NLP family exhibits functional diversity, as demonstrated by the genetic manipulation of NLPs in many microorganisms including F. oxysporum [15], B. cinerea [17], Magnaporthe oryzae [51], B. elliptica [52], and Diplodia seriata [53]. ...
The fungus Stemphylium lycopersici (S. lycopersici) is an economically important plant pathogen that causes grey leaf spot disease in tomato. However, functional genomic studies in S. lycopersici are lacking, and the factors influencing its pathogenicity remain largely unknown. Here, we present the first example of genetic transformation and targeted gene replacement in S. lycopersici. We functionally analyzed the NLP gene, which encodes a necrosis- and ethylene-inducing peptide 1 (Nep1)-like protein (NLP). We found that targeted disruption of the NLP gene in S. lycopersici significantly compromised its virulence on tomato. Moreover, our data suggest that NLP affects S. lycopersici conidiospore production and weakly affects its adaptation to osmotic and oxidative stress. Interestingly, we found that NLP suppressed the production of reactive oxygen species (ROS) in tomato leaves during S. lycopersici infection. Further, expressing the fungal NLP in tomato resulted in constitutive transcription of immune-responsive genes and inhibited plant growth. Through gene manipulation, we demonstrated the function of NLP in S. lycopersici virulence and development. Our work provides a paradigm for functional genomics studies in a non-model fungal pathogen system.
... (a) The toxins hypothesis. Several studies support the involvement of toxic metabolites of fungal origin (Bruno and Sparapano, 2006;Bruno et al., 2007;Andolfi et al., 2011;Schilling et al., 2021), a well-known mechanism in two major GTDs (i.e., Eutypa dieback and Botryosphaeria dieback; Colrat et al., 1999;Masi et al., 2018;Trotel-Aziz et al., 2019;Cobos et al., 2019;Schilling et al., 2021). Still, the evidence provided to explain the leaf stripe symptom development and yearly fluctuation in esca-affected vines remains unsatisfactory. ...
A peculiar symptom that may develop in grapevines affected by wood pathogens involved in the esca complex of diseases is the leaf stripe symptom, which also gives the name to the Grapevine Leaf Stripe Disease. Multiple studies have revealed strong links between fungal presence, wood symptomatology and expression of the leaf stripe symptom. However, numerous other factors have been shown to play roles in symptom onset, incidence, severity and yearly fluctuation of this disease. While the factors triggering the leaf stripe symptom are still under investigation, three control strategies have been proven effective for substantially reducing its expression, namely trunk surgery, and applications of sodium arsenite or a fertilizer mixture. These control strategies are examined here, including their (putative or confirmed) modes of action, and how they may influence the leaf stripe symptom development. In this article, we also propose the ‘edge’ hypothesis to tentatively explain symptoms onset, keeping in consideration past knowledge and recent advances in the understanding of the esca leaf stripe symptom. Ultimately, it is our intention to offer food-for-thought and stimulate debate within the phytopathological community.
... Neofusicoccum parvum-Bt67 caused BD symptoms on the rootlings of the two grapevine cultivars, as shoot full dieback, canker external necrosis, and shoot internal necrosis. Interestingly, the full dieback symptoms were more severe on Tempranillo than on Chardonnay (i.e., 37.5 and 28%, respectively, Figures 2D,G), suggesting a greater susceptibility to BD for Tempranillo than Chardonnay, as already reported by Luque et al. (2009) andCobos et al. (2019). Although there is a lack of comparative data between cultivars, the distinct susceptibility of some cultivars to GTDs has already been reported (Travadon et al., 2013;Fontaine et al., 2016b;Chacon et al., 2020;Reveglia et al., 2021), even within a same cultivar from one region to another or depending on the vintage (Mimiague and Le Gall, 1994). ...
... Since Botryosphaeriaceae are known to specifically metabolize grapevine phytoalexins (Stempien et al., 2017), which benefits pathogen fitness, we could suggest that the SA stimulation of the phenylpropanoid pathway and derivatives would wrongly serve the plant. In the case of Tempranillo exposed to Botryosphaeriaceae, the high constitutive expression of SA-dependent defense genes could thus appear as a disadvantage, confirming that Tempranillo would be less tolerant than Chardonnay to BD, as already reported by Luque et al. (2009) andCobos et al. (2019). Fortunately, in the Tempranillo pretreated with both BCAs, the expressions of genes PR1, PAL, and STS were repressed, and in the Tempranillo pretreated with Ta SC1 alone, the expression of the genes PAL and STS were repressed. ...
Grapevine trunk diseases (GTDs) are a big threat for global viticulture. Without effective
chemicals, biocontrol strategies are developed as alternatives to better cope with
environmental concerns. A combination of biological control agents (BCAs) could even
improve sustainable disease management through complementary ways of protection.
In this study, we evaluated the combination of Bacillus subtilis (Bs) PTA-271 and
Trichoderma atroviride (Ta) SC1 for the protection of Chardonnay and Tempranillo
rootlings against Neofusicoccum parvum Bt67, an aggressive pathogen associated
to Botryosphaeria dieback (BD). Indirect benefits offered by each BCA and their
combination were then characterized in planta, as well as their direct benefits in vitro.
Results provide evidence that (1) the cultivar contributes to the beneficial effects of Bs
PTA-271 and Ta SC1 against N. parvum, and that (2) the in vitro BCA mutual antagonism
switches to the strongest fungistatic effect toward Np-Bt67 in a three-way confrontation
test. We also report for the first time the beneficial potential of a combination of BCA
against Np-Bt67 especially in Tempranillo. Our findings highlight a common feature for
both cultivars: salicylic acid (SA)-dependent defenses were strongly decreased in plants
protected by the BCA, in contrast with symptomatic ones. We thus suggest that (1)
the high basal expression of SA-dependent defenses in Tempranillo explains its highest
susceptibility to N. parvum, and that (2) the cultivar-specific responses to the beneficial
Bs PTA-271 and Ta SC1 remain to be further investigated.
... Ascaulitoxin aglycone is one of three metabolites with herbicidal activity produced by Ascochyta caulina, and it is also a potential herbicide of Chenopodium album (Huffaker et al., 2011). Tentoxin, a powerful toxin produced by the plant pathogen Alternaria alternata, is a cyclic tetrapeptide compound (Lou et al., (Lara-Márquez et al., 2011;Zhang et al., 2014;Gomes et al., 2015;Guerriero et al., 2015;Li et al., 2017;Tan and Oliver, 2017;Proctor et al., 2018;Abro et al., 2019;Azhar et al., 2019;Cobos et al., 2019). CWBE, cell wall-degrading enzymes; PG, pectin polygalacturonase; PE, pectin methylesterase; EG, Endo-1,4-β-D-glucanase; PA, protease; CHI, chitinase; GR, growth regulator; AUX, auxin; CKs, cytokinins; BRs, brassinolides; ABA, abscisic acid; GA, gibberellic acid; SA, salicylic acid; JA, jasmonic acid; ET, ethylene; EP, effector proteins; FV, fungal virus; DV, DNA virus; RV, RNA virus. ...
... The interaction between plant-pathogenic fungi and the host plant can be understood as the interaction between the fungal Avr gene and the host plant R gene (Qin et al., 2018). Recognition of the Avr gene product by the R gene product results in incompatibility, such that the plant does not become diseased; when the R gene product does not recognize the Avr gene product, compatibility with the host plant results in disease (Figure 3) (Kobayashi et al., 2018;Cobos et al., 2019;Yu et al., 2019). The virulence effects and transport molecular mechanisms of effector proteins are still in the initial stages of research. ...
... In addition to the direct lethal effects of locust microspores, the sublethal effects, such as inhibition of the growth and development of locusts, egg laying, and gathering behavior, are perhaps more important. Locust microspores can FIGURE 3 | Interaction pattern between phytopathogenic fungi and plants (Pieterse et al., 2009;Cobos et al., 2019). (A) Upon pathogen attack, PAMPs activate PRRs in the host, resulting in a downstream signaling cascade that leads to PTI. (B) Virulent pathogens have acquired effectors (blue ellipses) that suppress PTI, resulting in ETS. ...
Phytopathogenic fungi decrease crop yield and quality and cause huge losses in agricultural production. To prevent the occurrence of crop diseases and insect pests, farmers have to use many synthetic chemical pesticides. The extensive use of these pesticides has resulted in a series of environmental and ecological problems, such as the increase in resistant weed populations, soil compaction, and water pollution, which seriously affect the sustainable development of agriculture. This review discusses the main advances in research on plant-pathogenic fungi in terms of their pathogenic factors such as cell wall-degrading enzymes, toxins, growth regulators, effector proteins, and fungal viruses, as well as their application as biocontrol agents for plant pests, diseases, and weeds. Finally, further studies on plant-pathogenic fungal resources with better biocontrol effects can help find new beneficial microbial resources that can control diseases.
... Some AEPs are considered as toxins, called necrosis-inducing proteins (NLPs), able to cause cell death. NLPs were first identified from culture filtrate of Fusarium oxysporum but have been isolated in oomycetes, fungi and bacteria, and have the ability to induce cell death and ethylene accumulation in plants (Gijzen and Nürnberger, 2006;Cobos et al., 2019). The structure of NLPs is remarkably conserved among long phylogenetic distance, from bacteria to oomycetes (Feng et al., 2014;Ottmann et al., 2009). ...
Oomycetes are eukaryote pathogens able to infect plants and animals. During host interaction, oomycetes secrete various molecules, named effectors, to counteract plant defence and modulate plant immunity. Crinklers (CRNs) and RxLR proteins represent the two main classes of cytoplasmic effectors described in oomycetes to date. Most of these effectors have not been yet characterized. In the root rot pathogen of legumes Aphanomyces euteiches, only the CRNs are present. Based on a previous study reported by our research group, we published an opinion paper focused on the emergence of DNA damaging effectors and their role during infection. Previous experiments indicated that one of these Crinklers, AeCRN5, harboured a functional translocation domain and dramatically disturbed root development. Here we reveal that AeCRN5 binds to RNA and interferes with biogenesis of various small RNAs, implicated in defence mechanisms or plant development. Additionally, comparative genetic analyses revealed a new class of putative effectors specific to Aphanomyces euteiches, composed by a large repertoire of small-secreted protein coding genes (SSP). Preliminary results on these SSPs point out that AeSSP1256 enhances host susceptibility. Functional characterisation of AeSSP1256 evidenced that this effector binds to RNA, relocalizes a plant RNA helicase and interferes with its activity, causing stress on plant ribosome biogenesis. This work highlights that various effector target nucleic acids and reveals that two effectors from distinct family are able to interact with plant RNA in order to interfere with RNA related defence mechanisms and plant development to promote pathogen infection.
