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.

A chromosome-scale assembly of Brassica carinata (BBCC) accession HC20 containing resistance to multiple pathogens and an early generation assessment of introgressions into B. juncea (AABB).

Brassica carinata (BBCC) commonly referred to as Ethiopian mustard is a natural allotetraploid containing the genomes of Brassica nigra (BB) and Brassica oleracea (CC). It is an oilseed crop endemic to the northeastern regions of Africa. Although it is under limited cultivation, B. carinata is valuable as it is resistant/highly tolerant to most of the pathogens affecting widely cultivated Brassica species of the U's triangle. We report a chromosome-scale genome assembly of B. carinata accession HC20 using long-read Oxford Nanopore sequencing and Bionano optical maps. The assembly has a scaffold N50 of ~39.8 Mb and covers ~1.11 Gb of the genome. We compared the long-read genome assemblies of the U's triangle species and found extensive gene collinearity between the diploids and allopolyploids with no evidence of major gene losses. Therefore, B. juncea (AABB), B. napus (AACC), and B. carinata can be regarded as strict allopolyploids. We cataloged the nucleotide-binding and leucine-rich repeat immune receptor (NLR) repertoire of B. carinata and, identified 465 NLRs, and compared these with the NLRs in the other Brassica species. We investigated the extent and nature of early-generation genomic interactions between the constituent genomes of B. carinata and B. juncea in interspecific crosses between the two species. Besides the expected recombination between the constituent B genomes, extensive homoeologous exchanges were observed between the A and C genomes. Interspecific crosses, therefore, can be used for transferring disease resistance from B. carinata to B. juncea and broadening the genetic base of the two allotetraploid species.

Transcriptome Analysis of the Necrotrophic Pathogen Alternaria brassicae Reveals Insights into Its Pathogenesis in Brassica juncea.

Alternaria blight or leaf spot caused by Alternaria brassicae has an enormous economic impact on the Brassica crops grown worldwide. Although the genome of A. brassicae has been sequenced, little is known about the genes that play a role during the infection of the host species. In this study, the transcriptome expression profile of A. brassicae during growth and infection was determined. Differential expression analysis revealed that 4,430 genes were differentially expressed during infection. Weighted gene coexpression network analysis helped identify 10 modules, which were highly correlated with growth and infection. Subsequent gene ontology (GO) enrichment analysis of the modules highlighted the involvement of biological processes such as toxin metabolism, ribosome biogenesis, polysaccharide catabolism, copper ion transport, and vesicular trafficking during infection. Additionally, 200 carbohydrate-active enzymes (CAZymes) and 80 potential effectors were significantly upregulated during infection. Furthermore, 18 secondary metabolite gene clusters were also differentially expressed during infection. The clusters responsible for the production of destruxin B, brassicicene C, and HC-toxin were significantly upregulated during infection. Collectively, these results provide an overview of the critical pathways underlying the pathogenesis of A. brassicae and highlight the distinct gene networks that are temporally regulated. The study thus provides novel insights into the transcriptional plasticity of a necrotrophic pathogen during infection of its host. Additionally, the in planta expression evidence for many potential effectors provides a theoretical basis for further investigations into the effector biology of necrotrophic pathogens such as A. brassicae. IMPORTANCE Alternaria brassicae is a necrotrophic pathogen that can infect almost all members of the Brassicaceae family. A. brassicae causes extensive yield losses in oilseed mustard and has practically restricted the cultivation of oilseed brassicas in regions with cool and foggy climatic conditions (foothills and mountainous terrains) where the severity of the pathogen is the highest. In this study, I identified the differentially expressed genes associated with the pathogenicity of A. brassicae through transcriptome sequencing. Also, I have been able to delineate pathways that are active during the early and late stages of infection. Consequently, this study has provided crucial insights into the molecular mechanisms underlying the pathogenesis of A. brassicae, an important necrotrophic pathogen.

Intrinsically Disordered Kiwellin Protein-Like Effectors Target Plant Chloroplasts and are Extensively Present in Rust Fungi.

The effector proteins produced by plant pathogens are one of the essential components of host-pathogen interaction. Despite being important, most of the effector proteins remain unexplored due to the diversity in their primary sequence generated by the high selection pressure of the host immune system. However to maintain the primary function in the infection process, these effectors may tend to maintain their native protein fold to perform the corresponding biological function. In the present study, unannotated candidate secretory effector proteins of sixteen major plant fungal pathogens were analyzed to find the conserved known protein folds using homology, ab initio, and Alpha Fold/Rosetta Fold protein dimensional (3D) structure approaches. Several unannotated candidate effector proteins were found to match various known conserved protein families potentially involved in host defense manipulation in different plant pathogens. Surprisingly a large number of plant Kiwellin proteins fold like secretory proteins (> 100) were found in studied rust fungal pathogens. Many of them were predicted as potential effector proteins. Furthermore, template independent modelling using Alpha Fold/Rosetta Fold analysis and structural comparison of these candidates also predicted them to match with plant Kiwellin proteins. We also found plant Kiwellin matching proteins outside rusts including several non-pathogenic fungi suggesting the broad function of these proteins. One of the highest confidently modeled Kiwellin matching candidates effectors, Pstr_13960 (97.8%), from the Indian P. striiformis race Yr9 was characterized using overexpression, localization, and deletion studies in Nicotiana benthamiana. The Pstr_13960 suppressed the BAX-induced cell death and localized in the chloroplast. Furthermore, the expression of the Kiwellin matching region (Pst_13960_kiwi) alone suppressed the BAX-induced cell death in N. benthamiana despite the change of location to the cytoplasm and nucleus, suggesting the novel function of the Kiwellin core fold in rust fungi. Molecular docking showed that Pstr_13960 can interact with plant Chorismate mutases (CMs) using three loops conserved in plant and rust Kiwellins. Further analysis of Pstr_13960 showed to contain Intrinsically disordered regions (IDRs) in place of the N-terminal ß1/ß2 region found in plant Kiwellins suggesting the evolution of rust Kiwellins-like effectors (KLEs). Overall, this study reports the presence of a Kiwellin protein-like fold containing a novel effector protein family in rust fungi depicting a classical example of the evolution of effectors at the structure level as Kiwellin effectors show very low significant similarity to plant Kiwellin at the sequence level.

Genome sequencing and assembly of Lathyrus sativus - a nutrient-rich hardy legume crop.

Grass pea (Lathyrus sativus) is a cool-season legume crop tolerant to drought, salinity, waterlogging, insects, and other biotic stresses. Despite these beneficial traits, this crop is not cultivated widely due to the accumulation of a neurotoxin - ß-N-oxalyl-L-α, ß-diaminopropionic acid (ß-ODAP) in the seeds and its association with neurolathyrism. In this study, we sequenced and assembled the genome of Lathyrus sativus cultivar Pusa-24, an elite Indian cultivar extensively used in breeding programs. The assembled genome of Lathyrus was 3.80 Gb in length, with a scaffold N50 of 421.39 Mb. BUSCO assessment indicated that 98.3% of highly conserved Viridiplantae genes were present in the assembly. A total of 3.17 Gb (83.31%) of repetitive sequences and 50,106 protein-coding genes were identified in the Lathyrus assembly. The Lathyrus genome assembly reported here thus provides a much-needed and robust foundation for various genetic and genomic studies in this vital legume crop.

Intrarectal Capsazepine Administration Modulates Colonic Mucosal Health in Mice.

Antagonism of transient receptor potential vanniloid-1 (TRPV1) and desensitization of transient receptor potential ankyrin-1 (TRPA1) nociceptors alleviate inflammatory bowel diseases (IBD)-associated chronic pain. However, there is limited literature available about their role in regulating the mucosal layer, its interaction with host physiology, and luminal microbial community. The present study focuses on the effects' intra rectal administration of capsazepine (modulator of TRPA1/TRPV1 expressing peptidergic sensory neurons) on colonic mucus production and gut health. We performed histological analysis, gut permeability alteration, gene expression changes, metabolite profiling, and gut microbial abundance in the ileum, colon, and cecum content of these animals. Intra rectal administration of capsazepine modulates TRPA1/TRPV1-positive nociceptors (behavioral pain assays) and resulted in damaged mucosal lining, increased gut permeability, and altered transcriptional profile of genes for goblet cell markers, mucus regulation, immune response, and tight junction proteins. The damage to mucosal lining prevented its role in enterosyne (short chain fatty acids) actions. These results suggest that caution must be exercised before employing TRPA1/TRPV1 modulation as a therapeutic option to alleviate pain caused due to IBD.

Functional Characterization of the Nep1-Like Protein Effectors of the Necrotrophic Pathogen - Alternaria brassicae.

Alternaria brassicae is an important necrotrophic pathogen that infects the Brassicaceae family. A. brassicae, like other necrotrophs, also secretes various proteinaceous effectors and metabolites that cause cell death to establish itself in the host. However, there has been no systematic study of A. brassicae effectors and their roles in pathogenesis. The availability of the genome sequence of A. brassicae in public domain has enabled the search for effectors and their functional characterization. Nep1-like proteins (NLPs) are a superfamily of proteins that induce necrosis and ethylene biosynthesis. They have been reported from a variety of microbes including bacteria, fungi, and oomycetes. In this study, we identified two NLPs from A. brassicae viz. AbrNLP1 and AbrNLP2 and functionally characterized them. Although both AbrNLPs were found to be secretory in nature, they localized differentially inside the plant. AbrNLP2 was found to induce necrosis in both host and non-host species, while AbrNLP1 could not induce necrosis in both species. Additionally, AbrNLP2 was shown to induce pathogen-associated molecular pattern (PAMP)-triggered immunity in both host and non-host species. Overall, our study indicates that AbrNLPs are functionally and spatially (subcellular location) distinct and may play different but important roles during the pathogenesis of A. brassicae.

Effector Biology of Biotrophic Plant Fungal Pathogens: Current Advances and Future Prospects.

The interaction of fungal pathogens with their host requires a novel invading mechanism and the presence of various virulence-associated components responsible for promoting the infection. The small secretory proteins, explicitly known as effector proteins, are one of the prime mechanisms of host manipulation utilized by the pathogen to disarm the host. Several effector proteins are known to translocate from fungus to the plant cell for host manipulation. Many fungal effectors have been identified using genomic, transcriptomic, and bioinformatics approaches. Most of the effector proteins are devoid of any conserved signatures, and their prediction based on sequence homology is very challenging, therefore by combining the sequence consensus based upon machine learning features, multiple tools have also been developed for predicting apoplastic and cytoplasmic effectors. Various post-genomics approaches like transcriptomics of virulent isolates have also been utilized for identifying active consortia of effectors. Significant progress has been made in understanding biotrophic effectors; however, most of it is underway due to their complex interaction with host and complicated recognition and signaling networks. This review discusses advances, and challenges in effector identification and highlighted various features of the potential effector proteins and approaches for understanding their genetics and strategies for regulation.

Anti-inflammatory Bifidobacterium strains prevent dextran sodium sulfate induced colitis and associated gut microbial dysbiosis in mice.

Crohn's and ulcerative colitis are common inflammatory conditions associated with Inflammatory bowel disease. Owing to the importance of diet based approaches for the prevention of inflammatory gut conditions, the present study was aimed to screen the human isolates of Bifidobacterium strains based on their ability to reduce LPS-induced inflammation in murine macrophage (RAW 264.7) cells and to evaluate prioritized strains for their preventive efficacy against ulcerative colitis in mice. Twelve out of 25 isolated strains reduced the production of LPS-induced nitric oxide and inflammatory cytokines. Furthermore, three strains, B. longum Bif10, B. breve Bif11, and B. longum Bif16 conferred protection against dextran sodium sulfate induced colitis in mice. The three strains prevented shortening of colon, spleen weight, percentage body weight change and disease activity index relative to colitis mice. Lower levels of Lipocalin-2, TNF-α, IL-1ß and IL-6 and improved SCFA levels were observed in Bifidobacterium supplemented mice relative to DSS counterparts. Bacterial composition of B. longum Bif10 and B. breve Bif11 fed mice was partly similar to the normal mice, while DSS and B. longum Bif16 supplemented mice showed deleterious alterations. At the genus level, Bifidobacterium supplementation inhibited the abundances of pathobionts such as Haemophilus, Klebsiella and Lachnospira there by conferring protection.

Linear and branched ß-Glucans degrading enzymes from versatile Bacteroides uniformis JCM 13288T and their roles in cooperation with gut bacteria.

ß-glucans are the dietary nutrients present in oats, barley, algae, and mushrooms. The macromolecules are well known for their immune-modulatory activity; however, how the human gut bacteria digest them is vaguely understood. In this study, Bacteroides uniformis JCM 13288 T was found to grow on laminarin, pustulan, and porphyran. We sequenced the genome of the strain, which was about 5.05 megabase pairs and contained 4868 protein-coding genes. On the basis of growth patterns of the bacterium, two putative polysaccharide utilization loci for ß-glucans were identified from the genome, and associated four putative genes were cloned, expressed, purified, and characterized. Three glycoside hydrolases (GHs) that were endo-acting enzymes (BuGH16, BuGH30, and BuGH158), and one which was an exo-acting (BuGH3) enzyme. The BuGH3, BuGH16, and BuGH158 can cleave linear exo/endo- ß- 1-3 linkages while BuGH30 can digest endo- ß- 1-6 linkages. BuGH30 and BuGH158 were further explored for their roles in digesting ß- glucans and generation of oligosaccharides, respectively. The BuGH30 predominately found to cleave long chain ß- 1-6 linked glucans, and obtained final product was gentiobiose. The BuGH158 used for producing oligosaccharides varying from degree of polymerization 2 to 7 from soluble curdlan. We demonstrated that these oligosaccharides can be utilized by gut bacteria, which either did not grow or poorly grew on laminarin. Thus, B. uniformis JCM 13288 T is not only capable of utilizing ß-glucans but also shares these glycans with human gut bacteria for potentially maintaining the gut microbial homeostasis.

Smut fungi as a stratagem to characterize rust effectors: opportunities and challenges.

The rust pathogens are one of the most complex fungi in the Basidiomycetes. The development of genomic resources for rust and other plant pathogens has opened the opportunities for functional genomics of fungal genes. Despite significant progress in the field of fungal genomics, functional characterization of the genome components has lacked, especially for the rust pathogens. Their obligate nature and lack of standard stable transformation protocol are the primary reasons for rusts to be one of the least explored genera despite its significance. In the recently sequenced rust genomes, a vast catalogue of predicted effectors and pathogenicity genes have been reported. However, most of these candidate genes remained unexplored due to the lack of suitable characterization methods. The heterologous expression of putative effectors in Nicotiana benthamiana and Arabidopsis thaliana has proved to be a rapid screening method for identifying the role of these effectors in virulence. However, no fungal system has been used for the functional validation of these candidate genes. The smuts, from the evolutionary point of view, are closely related to the rust pathogens. Moreover, they have been widely studied and hence could be a suitable model system for expressing rust fungal genes heterologously. The genetic manipulation methods for smuts are also well standardized. Complementation assays can be used for functional validation of the homologous genes present in rust and smut fungal pathogens, while the species-specific proteins can be expressed in the mutant strains of smut pathogens having reduced or no virulence for virulence analysis. We propose that smuts, especially Ustilago maydis, may prove to be a good model system to characterize rust effector proteins in the absence of methods to manipulate the rust genomes directly.

Draft Genome Sequence of Bifidobacterium pseudocatenulatum Bif4, Isolated from Healthy Infant Feces.

We report the 2.24-Mb draft genome sequence of Bifidobacterium pseudocatenulatum Bif4, isolated from a fecal sample from a healthy infant. The specific annotations revealed genes predictive of its probiotic attributes.

Correction to: Comparative genomics of Alternaria species provides insights into the pathogenic lifestyle of Alternaria brassicae - a pathogen of the Brassicaceae family.

Following the publication of this article [1], the authors reported that the captions of Figs. 2 and 3 were published in the incorrect order, whereby they mismatch with their corresponding images. The figures are reproduced in the correct sequence with the correct captions in this Correction article. The original article has been corrected.

Near-Complete Genome Assembly of Alternaria brassicae-A Necrotrophic Pathogen of Brassica Crops.

Alternaria brassicae, a necrotrophic fungal pathogen, causes Alternaria blight, an important disease of brassica crops. Although many Alternaria spp. have been sequenced, no genome information is available for A. brassicae, a monotypic lineage within the Alternaria genus. A highly contiguous genome assembly of A. brassicae has been generated using Nanopore MinION sequencing with an N50 of 2.98 Mb, yielding nine full chromosome-level sequences. This study adds to the current genomic resources available for the genus Alternaria and will provide opportunities for further analyses to unravel the mechanisms underlying pathogenicity of this important necrotrophic pathogen.

Comparative genomics of Alternaria species provides insights into the pathogenic lifestyle of Alternaria brassicae - a pathogen of the Brassicaceae family.

BACKGROUND: Alternaria brassicae, a necrotrophic pathogen, causes Alternaria Leaf Spot, one of the economically important diseases of Brassica crops. Many other Alternaria spp. such as A. brassicicola and A. alternata are known to cause secondary infections in the A. brassicae-infected Brassicas. The genome architecture, pathogenicity factors, and determinants of host-specificity of A. brassicae are unknown. In this study, we annotated and characterised the recently announced genome assembly of A. brassicae and compared it with other Alternaria spp. to gain insights into its pathogenic lifestyle. RESULTS: We also sequenced the genomes of two A. alternata isolates that were co-infecting B. juncea using Nanopore MinION sequencing for additional comparative analyses within the Alternaria genus. Genome alignments within the Alternaria spp. revealed high levels of synteny between most chromosomes with some intrachromosomal rearrangements. We show for the first time that the genome of A. brassicae, a large-spored Alternaria species, contains a dispensable chromosome. We identified 460 A. brassicae-specific genes, which included many secreted proteins and effectors. Furthermore, we have identified the gene clusters responsible for the production of Destruxin-B, a known pathogenicity factor of A. brassicae. CONCLUSION: The study provides a perspective into the unique and shared repertoire of genes within the Alternaria genus and identifies genes that could be contributing to the pathogenic lifestyle of A. brassicae.

Alternaria brassicae interactions with the model Brassicaceae member Arabidopsis thaliana closely resembles those with Mustard (Brassica juncea).

Alternaria leaf blight, a disease of oilseed Brassicas is caused by a necrotrophic phytopathogenic fungus Alternaria brassicae. The details of its pathogenesis and defence responses elicited in the host upon infection have not been thoroughly investigated. Here, Arabidopsis accession Gre-0 was identified to be highly susceptible to A. brassicae. A comparative histopathological analysis for disease progression and plant responses to A. brassicae in Arabidopsis and Brassica juncea revealed significant similarities between the two compatible pathosystems. Interestingly, in both the compatible hosts, ROS accumulation, cell death and callose deposition correlated with the development of the disease. Based on our results we propose that Arabidopsis-Alternaria brassicae can be an apt model pathosystem since it emulates the dynamics of the pathogen interaction with its natural host- Brassicas. The existing genetic diversity in Arabidopsis can be a starting point to screen for variation in responses to Alternaria leaf blight. Furthermore, several tools available for Arabidopsis can facilitate the dissection of genetic and molecular basis of resistance.

Genome-wide association mapping in Arabidopsis identifies novel genes underlying quantitative disease resistance to Alternaria brassicae.

Quantitative disease resistance (QDR) is the predominant form of resistance against necrotrophic pathogens. The genes and mechanisms underlying QDR are not well known. In the current study, the Arabidopsis-Alternaria brassicae pathosystem was used to uncover the genetic architecture underlying resistance to A. brassicae in a set of geographically diverse Arabidopsis accessions. Arabidopsis accessions revealed a rich variation in the host responses to the pathogen, varying from complete resistance to high susceptibility. Genome-wide association (GWA) mapping revealed multiple regions to be associated with disease resistance. A subset of genes prioritized on the basis of gene annotations and evidence of transcriptional regulation in other biotic stresses was analysed using a reverse genetics approach employing T-DNA insertion mutants. The mutants of three genes, namely At1g06990 (GDSL-motif lipase), At3g25180 (CYP82G1) and At5g37500 (GORK), displayed an enhanced susceptibility relative to the wild-type. These genes are involved in the development of morphological phenotypes (stomatal aperture) and secondary metabolite synthesis, thus defining some of the diverse facets of quantitative resistance against A. brassicae.

Genetic Architecture of Resistance to Alternaria brassicae in Arabidopsis thaliana: QTL Mapping Reveals Two Major Resistance-Conferring Loci.

Alternaria brassicae, a necrotrophic fungal pathogen, causes Alternaria blight, one of the most important diseases of oleiferous Brassica crops. The current study utilized Arabidopsis as a model to decipher the genetic architecture of defense against A. brassicae. Significant phenotypic variation that was largely genetically determined was observed among Arabidopsis accessions in response to pathogen challenge. Three biparental mapping populations were developed from three resistant accessions viz. CIBC-5, Ei-2, and Cvi-0 and two susceptible accessions - Gre-0 and Zdr-1 (commonly crossed to CIBC-5 and Ei-2). A total of six quantitative trait locus (QTLs) governing resistance to A. brassicae were identified, five of which were population-specific while one QTL was common between all the three mapping populations. Interestingly, the common QTL had varying phenotypic contributions in different populations, which can be attributed to the genetic background of the parental accessions. The presence of both common and population-specific QTLs indicate that resistance to A. brassicae is quantitative, and that different genes may mediate resistance to the pathogen in different accessions. Two of the QTLs had moderate-to-large effects, one of which explained nearly 50% of the variation. The large effect QTLs may therefore contain genes that could play a significant role in conferring resistance even in heterologous hosts.