Transcriptome responses of Arabidopsis to necrotrophic fungus Alternaria brassicae reveal pathways and candidate genes associated with resistance.

Alternaria leaf blight (ALB), caused by a necrotrophic fungus Alternaria brassicae is a serious disease of oleiferous Brassicas resulting in significant yield losses worldwide. No robust resistance against A. brassicae has been identified in the Brassicas. Natural accessions of Arabidopsis show a spectrum of responses to A. brassicae ranging from high susceptibility to complete resistance. To understand the molecular mechanisms of resistance/ susceptibility, we analysed the comparative changes in the transcriptome profile of Arabidopsis accessions with contrasting responses- at different time points post-infection. Differential gene expression, GO enrichment, pathway enrichment, and weighted gene co-expression network analysis (WGCNA) revealed reprogramming of phenylpropanoid biosynthetic pathway involving lignin, hydroxycinnamic acids, scopoletin, anthocyanin genes to be highly associated with resistance against A. brassicae. T-DNA insertion mutants deficient in the biosynthesis of coumarin scopoletin exhibited enhanced susceptibility to A. brassicae. The supplementation of scopoletin to medium or exogenous application resulted in a significant reduction in the A. brassicae growth. Our study provides new insights into the transcriptome dynamics in A. brassicae-challenged Arabidopsis and demonstrates the involvement of coumarins in plant immunity against the Brassica pathogen A. brassicae.

Bioprospecting endophytic fungi for bioactive metabolites with seed germination promoting potentials.

There is an urgent need for new bioactive molecules with unique mechanisms of action and chemistry to address the issue of incorrect use of chemical fertilizers and pesticides, which hurts both the environment and the health of humans. In light of this, research was done for this work to isolate, identify, and evaluate the germination-promoting potential of various plant species' fungal endophytes. Zea mays L. (maize) seed germination was examined using spore suspension of 75 different endophytic strains that were identified. Three promising strains were identified through screening to possess the ability mentioned above. These strains Alternaria alternate, Aspergilus flavus, and Aspergillus terreus were isolated from the stem of Tecoma stans, Delonix regia, and Ricinus communis, respectively. The ability of the three endophytic fungal strains to produce siderophore and indole acetic acid (IAA) was also examined. Compared to both Aspergillus flavus as well as Aspergillus terreus, Alternaria alternata recorded the greatest rates of IAA, according to the data that was gathered. On CAS agar versus blue media, all three strains failed to produce siderophores. Moreover, the antioxidant and antifungal potentials of extracts from these fungi were tested against different plant pathogens. The obtained results indicated the antioxidant and antifungal activities of the three fungal strains. GC-Mass studies were carried out to determine the principal components in extracts of all three strains of fungi. The three strains' fungus extracts included both well-known and previously unidentified bioactive compounds. These results may aid in the development of novel plant growth promoters by suggesting three different fungal strains as sources of compounds that may improve seed germination. According to the study that has been given, as unexplored sources of bioactive compounds, fungal endophytes have great potential.

The Fungal Diversity and Potential Pathogens Associated with Postharvest Fruit Rot of 'Huangguan' Pear (Pyrus bretschneideri) in Hebei Province, China.

Postharvest fruit rot caused by pathogens is a serious problem in the pear industry. This study investigated the fungal diversity and main pathogens and identified a new pathogen in the stored 'Huangguan' pear (Pyrus bretschneideri Rehd.), the dominant pear variety in northern China. We sampled 20 refrigeration houses from five main producing regions in Hebei Province and used Illumina sequencing technology to detect the fungal composition. Alternaria (56.3%) was the most abundant fungus, followed by Penicillium (9.2%) and Monilinia (6.2%). We also isolated and identified nine strains of Alternaria and four strains of Penicillium. Moreover, we observed a new postharvest fruit disease in 'Huangguan' pear caused by Stemphylium eturmiunum, which was confirmed by phylogenetic analysis by combining the sequences of three conserved genes, including internal transcribed spacer, gapdh, and calmodulin. This study marks the first documentation of S. eturmiunum causing fruit rot in 'Huangguan' pears, offering valuable insights for identifying and controlling this newly identified postharvest disease.

Acibenzolar-S-methyl activates phenylpropanoid pathway to enhance resistance against Alternaria alternata in pear fruit.

BACKGROUND: Alternaria alternata is a causal agent of black spot rot of pear fruit after harvest. Acibenzolar-S-methyl (ASM) has been shown to be a potential elicitor of tolerance in several horticultural products. This work was performed to research the influence of ASM on black spot rot of Docteur Jules Guyot pears and vital enzyme activity and gene expression in the phenylpropanoid pathway. RESULTS: ASM remarkably decreased the lesion diameter of A. alternata-inoculated pears. ASM also increased phenylalanine ammonialyase, cinnamate 4-hydroxylase, cinnamyl alcohol dehydrogenase, peroxidase, polyphenol oxidase activities and gene expression, and enhanced 4-coumarate/coenzyme A ligase activity in pears. Moreover, ASM improved the content of phenylalanine, total phenolic compounds, caffeic acid, flavonoids, anthocyanin and lignin in pears. CONCLUSION: ASM could modulate vital enzyme activity and gene expression in the phenylpropanoid pathway to accelerate metabolite synthesis, thereby enhancing resistance against A. alternata in pears. © 2022 Society of Chemical Industry.

CCR -NB-LRR proteins MdRNL2 and MdRNL6 interact physically to confer broad-spectrum fungal resistance in apple (Malus × domestica).

Apple leaf spot, a disease caused by Alternaria alternata f. sp. mali and other fungal species, leads to severe defoliation and results in tremendous losses to the apple (Malus × domestica) industry in China. We previously identified three RPW8, nucleotide-binding, and leucine-rich repeat domain CCR -NB-LRR proteins (RNLs), named MdRNL1, MdRNL2, and MdRNL3, that contribute to Alternaria leaf spot (ALT1) resistance in apple. However, the role of NB-LRR proteins in resistance to fungal diseases in apple remains poorly understood. We therefore used MdRNL1/2/3 as baits to screen ALT1-inoculated leaves for interacting proteins and identified only MdRNL6 (another RNL) as an interactor of MdRNL2. Protein interaction assays demonstrated that MdRNL2 and MdRNL6 interact through their NB-ARC domains. Transient expression assays in apple indicated that complexes containing both MdRNL2 and MdRNL6 are necessary for resistance to Alternaria leaf spot. Intriguingly, the same complexes were also required to confer resistance to Glomerella leaf spot and Marssonina leaf spot in transient expression assays. Furthermore, stable transgenic apple plants with suppressed expression of MdRNL6 showed hypersensitivity to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot; these effects were similar to the effects of suppressing MdRNL2 expression in transgenic apple plantlets. The identification of these novel broad-spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple.

Alternaria alternata-induced airway epithelial signaling and inflammatory responses via protease-activated receptor-2 expression.

Inhalation of the fungus Alternaria alternata is associated with an increased risk of allergic asthma development and exacerbations. Recent work in acute exposure animal models suggests that A. alternata-induced asthma symptoms, which include inflammation, mucus overproduction and airway hyperresponsiveness, are due to A. alternata proteases that act via protease-activated receptor-2 (PAR2). However, because other active components present in A. alternata may be contributing to asthma pathophysiology through alternative signaling, the specific role PAR2 plays in asthma initiation and maintenance remains undefined. Airway epithelial cells provide the first encounter with A. alternata and are thought to play an important role in initiating the physiologic response. To better understand the role for PAR2 airway epithelial signaling we created a PAR2-deficient human bronchial epithelial cell line (16HBEPAR-/-) from a model bronchial parental line (16HBE14o-). Comparison of in vitro physiologic responses in these cell lines demonstrated a complete loss of PAR2 agonist (2at-LIGRL-NH2) response and significantly attenuated protease (trypsin and elastase) and A. alternata responses in the 16HBEPAR-/- line. Apical application of A. alternata to 16HBE14o- and 16HBEPAR2-/- grown at air-liquid interface demonstrated rapid, PAR2-dependent and independent, inflammatory cytokine, chemokine and growth factor basolateral release. In conclusion, the novel human PAR2-deficient cell line allows for direct in vitro examination of the role(s) for PAR2 in allergen challenge with polarized human airway epithelial cells.

Identification of effector CEP112 that promotes the infection of necrotrophic Alternaria solani.

BACKGROUND: Alternaria solani is a typical necrotrophic pathogen that can cause severe early blight on Solanaceae crops and cause ring disease on plant leaves. Phytopathogens produce secretory effectors that regulate the host immune response and promote pathogenic infection. Effector proteins, as specialized secretions of host-infecting pathogens, play important roles in disrupting host defense systems. At present, the role of the effector secreted by A. solani during infection remains unclear. We report the identification and characterization of AsCEP112, an effector required for A. solani virulence. RESULT: The AsCEP112 gene was screened from the transcriptome and genome of A. solani on the basis of typical effector signatures. Fluorescence quantification and transient expression analysis showed that the expression level of AsCEP112 continued to increase during infection. The protein localized to the cell membrane of Nicotiana benthamiana and regulated senescence-related genes, resulting in the chlorosis of N. benthamiana and tomato leaves. Moreover, comparative analysis of AsCEP112 mutant obtained by homologous recombination with wild-type and revertant strains indicated that AsCEP112 gene played an active role in regulating melanin formation and penetration in the pathogen. Deletion of AsCEP112 also reduced the pathogenicity of HWC-168. CONCLUSION: Our findings demonstrate that AsCEP112 was an important effector protein that targeted host cell membranes. AsCEP112 regulateed host senescence-related genes to control host leaf senescence and chlorosis, and contribute to pathogen virulence.

Integrated transcriptomic and transgenic analyses reveal potential mechanisms of poplar resistance to Alternaria alternata infection.

BACKGROUND: Populus davidiana × P. bollena is a species of poplar from northeastern China that is characterized by cold resistance and fast growth but now suffers from pathogen infections. Leaf blight caused by Alternaria alternata has become a common poplar disease that causes serious economic impacts, but the molecular mechanisms of resistance to A. alternata in P. davidiana × P. bollena are still unclear. RESULTS: In this study, the transcriptomic response of P. davidiana × P. bollena to A. alternata infection was determined via RNA-Seq. Twelve cDNA libraries were generated from RNA isolated from three biological replicates at four time points (0, 2, 3, and 4 d post inoculation), and a total of 5,930 differentially expressed genes (DEGs) were detected (| log2 fold change |≥ 1 and FDR values < 0.05). Functional analysis revealed that the DEGs were mainly enriched for the "plant hormone signal transduction" pathway, followed by the "phenylpropanoid biosynthesis" pathway. In addition, DEGs that encode defense-related proteins and are related to ROS metabolism were also identified. Numerous transcription factors, such as the bHLH, WRKY and MYB families, were also induced by A. alternata infection. Among these DEGs, those related to JA biosynthesis and JA signal transduction were consistently activated. Therefore, the lipoxygenase gene PdbLOX2, which is involved in JA biosynthesis, was selected for functional characterization. Overexpression of PdbLOX2 enhanced the resistance of P. davidiana × P. bollena to A. alternata, whereas silencing this gene enhanced susceptibility to A. alternata infection. CONCLUSIONS: These results provide new insight into the molecular mechanisms of poplar resistance to A. alternata infection and provide candidate genes for breeding resistant cultivars using genetic engineering.

The elemental defense effect of cadmium on Alternaria brassicicola in Brassica juncea.

BACKGROUND: The elemental defense hypothesis states a new defensive strategy that hyperaccumulators defense against herbivores or pathogens attacks by accumulating heavy metals. Brassica juncea has an excellent ability of cadmium (Cd) accumulation. However, the elemental defense effect and its regulation mechanism in B. juncea remain unclear. RESULTS: In this study, we profiled the elemental defense effect and the molecular regulatory mechanism in Cd-accumulated B. juncea after Alternaria brassicicola infection. B. juncea treated with 180 mg Kg- 1 DW CdCl2 2.5H2O exhibited obvious elemental defense effect after 72 h of infection with A. brassicicola. The expression of some defense-related genes including BjNPR1, BjPR12, BjPR2, and stress-related miRNAs (miR156, miR397, miR398a, miR398b/c, miR408, miR395a, miR395b, miR396a, and miR396b) were remarkably elevated during elemental defense in B. juncea. CONCLUSIONS: The results indicate that Cd-accumulated B. juncea may defend against pathogens by coordinating salicylic acid (SA) and jasmonic acid (JA) mediated systemic acquired resistance (SAR) and elemental defense in a synergistic joint effect. Furthermore, the expression of miRNAs related to heavy metal stress response and disease resistance may regulate the balance between pathogen defense and heavy metal stress-responsive in B. juncea. The findings provide experimental evidence for the elemental defense hypothesis in plants from the perspectives of phytohormones, defense-related genes, and miRNAs.

Regulation of Eosinophil Recruitment and Allergic Airway Inflammation by Tropomyosin Receptor Kinase A.

Eosinophilia is a hallmark of allergic airway inflammation (AAI). Identifying key molecules and specific signaling pathways that regulate eosinophilic inflammation is critical for development of novel therapeutics. Tropomycin receptor kinase A (TrkA) is the high-affinity receptor for nerve growth factor. AAI is associated with increased expression of TrkA by eosinophils; however, the functional role of TrkA in regulating eosinophil recruitment and contributing to AAI is poorly understood. This study identifies, to our knowledge, a novel mechanism of eotaxin-mediated activation of TrkA and its role in regulating eosinophil recruitment by using a chemical-genetic approach to specifically inhibit TrkA kinase activity with 1-NM-PP1 in TrkAF592A-knock-in (TrkA-KI) eosinophils. Blockade of TrkA by 1-NM-PP1 enhanced eosinophil spreading on VCAM-1 but inhibited eotaxin-1 (CCL11)-mediated eosinophil migration, calcium flux, cell polarization, and ERK1/2 activation, suggesting that TrkA is an important player in the signaling pathway activated by eotaxin-1 during eosinophil migration. Further, blockade of matrix metalloprotease with BB-94 inhibited eotaxin-1-induced TrkA activation and eosinophil migration, additively with 1-NM-PP1, indicating a role for matrix metalloproteases in TrkA activation. TrkA inhibition in Alternaria alternata-challenged TrkA-KI mice markedly inhibited eosinophilia and attenuated various features of AAI. These findings are indicative of a distinctive eotaxin-mediated TrkA-dependent signaling pathway, which, in addition to other TrkA-activating mediators, contributes to eosinophil recruitment during AAI and suggests that targeting the TrkA signaling pathway to inhibit eosinophil recruitment may serve as a therapeutic strategy for management of eosinophilic inflammation in allergic airway disease, including asthma.

Comparative Analysis of Alternative Splicing in Two Contrasting Apple Cultivars Defense against Alternaria alternata Apple Pathotype Infection.

Alternaria blotch disease, caused by the Alternaria alternata apple pathotype (A. alternata AP), is one of the most serious fungal diseases in apples. Alternative splicing (AS), one of the pivotal post-transcriptional regulatory mechanisms, plays essential roles in various disease resistance responses. Here, we performed RNA-Seq for two apple cultivars (resistant cultivar 'Jonathan' (J) and susceptible cultivar 'Starking Delicious' (SD)) infected by A. alternata AP to further investigate their AS divergence. In total, 1454, 1780, 1367 and 1698 specifically regulated differential alternative splicing (DAS) events were detected in J36, J72, SD36 and SD72 groups, respectively. Retained intron (RI) was the dominant AS pattern. Conformably, 642, 764, 585 and 742 uniquely regulated differentially spliced genes (DSGs) were found during A. alternata AP infection. Comparative analysis of AS genes in differential splicing and expression levels suggested that only a small proportion of DSGs overlapped with differentially expressed genes (DEGs). Gene ontology (GO) enrichment analysis demonstrated that the DSGs were significantly enriched at multiple levels of gene expression regulation. Briefly, the specific AS was triggered in apple defense against A. alternata AP. Therefore, this study facilitates our understanding on the roles of AS regulation in response to A. alternata AP infection in apples.

Metapopulation Structure Predicts Population Dynamics in the Cakile maritima-Alternaria brassicicola Host-Pathogen Interaction.

AbstractIn symbiotic interactions, spatiotemporal variation in the distribution or population dynamics of one species represents spatial and temporal heterogeneity of the landscape for the other. Such interdependent demographic dynamics result in situations where the relative importance of biotic and abiotic factors in determining ecological processes is complicated to decipher. Using a detailed survey of three metapopulations of the succulent plant Cakile maritima and the necrotrophic fungus Alternaria brassicicola located along the southeastern Australian coast, we developed a series of statistical analyses-namely, synchrony analysis, patch occupancy dynamics, and a spatially explicit metapopulation model-to understand how habitat quality, weather conditions, dispersal, and spatial structure determine metapopulation dynamics. Climatic conditions are important drivers, likely explaining the high synchrony among populations. Host availability, landscape features facilitating dispersal, and habitat conditions also impact the occurrence and spread of disease. Overall, we show that the collection of extensive data on host and pathogen population dynamics, in combination with spatially explicit epidemiological modeling, makes it possible to accurately predict disease dynamics-even when there is extreme variability in host population dynamics. Finally, we discuss the importance of genetic information for predicting demographic dynamics in this pathosystem.

Antibacterial and antioxidant activities of endophytic fungi and nettle (Urtica dioica L.) leaves as their host.

Nettle (Urtica dioica L), as a plant rich in biologically active compounds, is one of the most important plants used in herbal medicine. Studies have shown that this plant has antioxidant, antiplatelet, hypoglycemic and hypocholesterolemia effects. In this study, we characterized three Alternaria endophytic fungi isolated from their host U. dioica. We hypothesized that these endophytic fungi can produce new bioactive metabolites, which may possess the bioactive property with potential application in the medical and pharmaceutical industries. The antibacterial activity was evaluated against reference and isolated strains, including Methicillin-Resistant Staphylococcus aureus. A wide range of antimicrobial activities similar to those measured in nettle leaves was detected especially for Alternaria sorghi. Furthermore, the highest antioxidant activity detected with DPPH free radical scavenging was measured for A. sorghi and nettle leaves ethyl acetate extracts. In addition, whereas catalase activity was similar in the three isolated fungi and nettle leaves, total thiol content and superoxide dismutase activity were significantly higher in leaves. A. sorghi showed the best activities compared to other isolated fungi. The characterization and further production of bioactive compounds produced by this endophyte should be investigated to fight bacteria and especially those that develop drug multi-resistance.

Overexpression of an apple LysM-containing protein gene, MdCERK1-2, confers improved resistance to the pathogenic fungus, Alternaria alternata, in Nicotiana benthamiana.

BACKGROUND: Lysin motif (LysM)-containing proteins are involved in the recognition of fungal and bacterial pathogens. However, few studies have reported on their roles in the defense responses of woody plants against pathogens. A previous study reported that the apple MdCERK1 gene was induced by chitin and Rhizoctonia solani, and its protein can bind to chitin. However, its effect on defense responses has not been investigated. RESULTS: In this study, a new apple CERK gene, designated as MdCERK1-2, was identified. It encodes a protein that shares high sequence identity with the previously reported MdCERK1 and AtCERK1. Its chitin binding ability and subcellular location are similar to MdCERK1 and AtCERK1, suggesting that MdCERK1-2 may play a role in apple immune defense responses as a pattern recognition receptor (PRR). MdCERK1-2 expression in apple was induced by 2 fungal pathogens, Botryosphaeria dothidea and Glomerella cingulate, but not by the bacterial pathogen, Erwinia amylovora, indicating that MdCERK1-2 is involved in apple anti-fungal defense responses. Further functional analysis by heterologous overexpression (OE) in Nicotiana benthamiana (Nb) demonstrated that MdCERK1-2 OE improved Nb resistance to the pathogenic fungus, Alternaria alternata. H2O2 accumulation and callose deposition increased after A. alternata infection in MdCERK1-2 OE plants compared to wild type (WT) and empty vector (EV)-transformed plants. The induced expression of NbPAL4 by A. alternata significantly (p < 0.01, n = 4) increased in MdCERK1-2 OE plants. Other tested genes, including NbNPR1, NbPR1a, NbERF1, and NbLOX1, did not exhibit significant changes after A. alternata infection in OE plants compared to EV or WT plants. OE plants also accumulated more polyphenols after A. alternata infection. CONCLUSIONS: Heterologous MdCERK1-2 OE affects multiple defense responses in Nb plants and increased their resistance to fungal pathogens. This result also suggests that MdCERK1-2 is involved in apple defense responses against pathogenic fungi.

Prior exposure of Arabidopsis seedlings to mechanical stress heightens jasmonic acid-mediated defense against necrotrophic pathogens.

BACKGROUND: Prolonged mechanical stress (MS) causes thigmomorphogenesis, a stress acclimation response associated with increased disease resistance. What remains unclear is if; 1) plants pre-exposed to a short period of repetitive MS can prime defence responses upon subsequent challenge with necrotrophic pathogens, 2) MS mediates plant immunity via jasmonic acid (JA) signalling, and 3) a short period of repetitive MS can cause long-term changes in gene expression resembling a stress-induced memory. To address these points, 10-days old juvenile Arabidopsis seedlings were mechanically stressed for 7-days using a soft brush and subsequently challenged with the necrotrophic pathogens, Alternaria brassicicola, and Botrytis cinerea. Here we assessed how MS impacted structural cell wall appositions, disease symptoms and altered gene expression in response to infection. RESULTS: The MS-treated plants exhibited enhanced cell wall appositions and jasmonic acid (JA) accumulation that correlated with a reduction in disease progression compared to unstressed plants. The expression of genes involved in JA signalling, callose deposition, peroxidase and phytoalexin biosynthesis and reactive oxygen species detoxification were hyper-induced 4-days post-infection in MS-treated plants. The loss-of-function in JA signalling mediated by the JA-insensitive coronatine-insensitive 1 (coi1) mutant impaired the hyper-induction of defense gene expression and promoted pathogen proliferation in MS-treated plants subject to infection. The basal expression level of PATHOGENESIS-RELATED GENE 1 and PLANT DEFENSIN 1.2 defense marker genes were constitutively upregulated in rosette leaves for 5-days post-MS, as well as in naïve cauline leaves that differentiated from the inflorescence meristem well after ceasing MS. CONCLUSION: This study reveals that exposure of juvenile Arabidopsis plants to a short repetitive period of MS can alter gene expression and prime plant resistance upon subsequent challenge with necrotrophic pathogens via the JA-mediated COI1 signalling pathway. MS may facilitate a stress-induced memory to modulate the plant's response to future stress encounters. These data advance our understanding of how MS primes plant immunity against necrotrophic pathogens and how that could be utilised in sustainable agricultural practices.

A novel mycovirus isolated from the plant-pathogenic fungus Alternaria dianthicola.

Here, we describe the molecular characterization of a novel mycovirus isolated from a phytopathogenic fungus, Alternaria dianthicola, which we have named "Alternaria dianthicola dsRNA virus 1" (AdRV1). AdRV1 has a genome of 3,014 bp that contains two non-overlapping open reading frames (ORF1 and 2) coding for a hypothetical protein and an RNA-dependent RNA polymerase (RdRp), respectively. Based on the RdRp, AdRV1 is phylogenetically related to some unclassified dsRNA mycoviruses, including Alternaria longipes dsRNA virus 1, and shows a distant relationship to members of the family Partitiviridae. To the best of our knowledge, this is the first report of mycovirus infecting A. dianthicola.

Redox-Dependent Structural Modification of Nucleoredoxin Triggers Defense Responses against Alternaria brassicicola in Arabidopsis.

In plants, thioredoxin (TRX) family proteins participate in various biological processes by regulating the oxidative stress response. However, their role in phytohormone signaling remains largely unknown. In this study, we investigated the functions of TRX proteins in Arabidopsis thaliana. Quantitative polymerase chain reaction (qPCR) experiments revealed that the expression of ARABIDOPSIS NUCLEOREDOXIN 1 (AtNRX1) is specifically induced by the application of jasmonic acid (JA) and upon inoculation with a necrotrophic fungal pathogen, Alternaria brassicicola. The AtNRX1 protein usually exists as a low molecular weight (LMW) monomer and functions as a reductase, but under oxidative stress AtNRX1 transforms into polymeric forms. However, the AtNRX1M3 mutant protein, harboring four cysteine-to-serine substitutions in the TRX domain, did not show structural modification under oxidative stress. The Arabidopsisatnrx1 null mutant showed greater resistance to A. brassicicola than wild-type plants. In addition, plants overexpressing both AtNRX1 and AtNRX1M3 were susceptible to A. brassicicola infection. Together, these findings suggest that AtNRX1 normally suppresses the expression of defense-responsive genes, as if it were a safety pin, but functions as a molecular sensor through its redox-dependent structural modification to induce disease resistance in plants.

Morpho-Pathological and Global Transcriptomic Analysis Reveals the Robust Nonhost Resistance Responses in Chickpea Interaction with Alternaria brassicae.

Alternaria blight, caused by Alternaria brassicae, causes considerable yield loss in Brassica crops. While several blight-resistant varieties have been developed using resistance sources from host germplasm, none of them are entirely successful in imparting durable resistance. This has prompted the exploration of novel gene pools of nonhost plant species. Nonhost resistance (NHR) is a durable form of resistance, comprising pre- and postinvasion layers of defense. We aimed to identify the molecular basis of NHR to A. brassicae and identify the layers of NHR operating in a nonhost, chickpea (Cicer arietinum). To elucidate the layers of NHR operating against A. brassicae, we compared the histopathology and infection patterns of A. brassicae in C. arietinum and Brassica juncea. Delayed conidial germination, impeded hyphal growth, suppressed appressorium formation, and limited hyphal penetration occurred in the nonhost plant compared with the host plant, implying the involvement of the preinvasion layer of NHR in C. arietinum. Next, we investigated the molecular basis of robust NHR, in C. arietinum challenged with A. brassicae, by microarray-based global transcriptome profiling. Genes involved in stomatal closure, cuticular wax biosynthesis, cell-wall modification, and secondary metabolite production (contributing to preinvasion NHR) as well as reactive oxygen species (ROS) and cell death (contributing to postinvasion NHR) were found to be upregulated. Consistent with transcriptomic analysis, the morpho-pathological analysis revealed stomatal closure, ROS accumulation, and localized cell death in C. arietinum as the defense strategies against A. brassicae. Thus, we identified NHR-contributing genes with potential applications in blight resistance gene transfer to B. juncea.

The Beneficial Root-Colonizing Fungus Mortierella hyalina Promotes the Aerial Growth of Arabidopsis and Activates Calcium-Dependent Responses That Restrict Alternaria brassicae-Induced Disease Development in Roots.

The endophytic fungus Mortierella hyalina colonizes the roots of Arabidopsis thaliana and stimulates growth and biomass production of the aerial parts but not of roots. An exudate fraction from the fungus induces rapid and transient cytoplasmic Ca2+elevation in the roots. The Ca2+ response does not require the well-characterized (co)receptors BAK1, CERK1, and FLS2 for pathogen-associated molecular patterns, and the Ca2+ channels GLR-2.4, GLR-2.5, and GLR-3.3 or the vacuolar TWO PORE CHANNEL1, which might be involved in cytoplasmic Ca2+ elevation. We isolated an ethyl-methane-sulfonate-induced Arabidopsis mutant that is impaired in this Ca2+ response. The roots of the mutant are impaired in M. hyalina-mediated suppression of immune responses after Alternaria brassicae infection, i.e., jasmonate accumulation, generation of reactive oxygen species, as well as the activation of jasmonate-related defense genes. Furthermore, they are more colonized by M. hyalina than wild-type roots. We propose that the mutant gene product is involved in a Ca2+-dependent signaling pathway activated by M. hyalina to suppress immune responses in Arabidopsis roots.

Enhancement of ABA Sensitivity Through Conditional Expression of the ARF10 Gene in Brassica juncea Reveals Fertile Plants with Tolerance Against Alternaria brassicicola.

Concomitant increase of auxin-responsive factors ARF16 and ARF17, along with enhanced expression of ARF10 in resistant Sinapis alba compared with that in susceptible Brassica juncea upon challenge with Alternaria brassicicola, revealed that abscisic acid (ABA)-auxin crosstalk is a critical factor for resistance response. Here, we induced the ABA response through conditional expression of ARF10 in B. juncea using the A. brassicicola-inducible GH3.3 promoter. Induced ABA sensitivity caused by conditional expression of ARF10 in transgenic B. juncea resulted in tolerance against A. brassicicola and led to enhanced expression of several ABA-responsive genes without affecting the auxin biosynthetic gene expression. Compared with ABI3 and ABI4, ABI5 showed maximum upregulation in the most tolerant transgenic lines upon pathogen challenge. Moreover, elevated expression of ARF10 by different means revealed a direct correlation between ARF10 expression and the induction of ABI5 protein in B. juncea. Through in vitro DNA-protein experiments and chromosome immunoprecipitation using the ARF10 antibody, we demonstrated that ARF10 interacts with the auxin-responsive elements of the ABI5 promoter. This suggests that ARF10 may function as a modulator of ABI5 to induce ABA sensitivity and mediate the resistance response against A. brassicicola.