Hybrid Genome Assembly Reveals Effector Arsenal of White Rust Pathogen in Indian Mustard

The oomycete pathogen Albugo candida, the causal agent of white rust disease, remains one of the most destructive biotrophic threats to oilseed brassicas worldwide. In a newly published study in Frontiers in Fungal Biology, a team of Indian researchers has delivered a high-quality hybrid genome assembly of a virulent Indian isolate of A. candida race Ac2V, the race responsible for devastating yield losses of up to 60 percent in Brassica juncea, commonly known as Indian mustard. By combining short-read Illumina sequencing with long-read Oxford Nanopore technology, the researchers have produced the most complete picture yet of the genomic architecture and secreted effector repertoire of this economically important pathogen under Indian agroclimatic conditions.

White rust disease earns its name from the characteristic creamish-white pustules that erupt on the underside of infected leaves, filled with masses of zoosporangia. As the disease progresses, these pustules coalesce into larger lesions, and in severe cases the infection spreads systemically to the floral structures, producing deformed inflorescences known as stagheads. The pathogen is an obligate biotroph, meaning it can only survive on living host tissue, and it has co-evolved with its brassica hosts to a remarkable degree. More than 24 physiological races of A. candida have been documented, collectively capable of infecting over 200 species across 63 genera, making the pathogen a formidable and adaptable foe for agricultural systems.

The research team, led by scientists at the ICAR-National Institute for Plant Biotechnology in New Delhi, began by maintaining a pure culture of the Ac2V Delhi isolate on susceptible B. juncea cv. Pusa Jaikisan plants under controlled growth chamber conditions. They collected zoosporangial powder from heavily infected leaves at 12 to 14 days post-inoculation and extracted high-molecular-weight genomic DNA using a modified CTAB protocol with cryogenic bead beating. The DNA was validated for purity through internal transcribed spacer (ITS) amplification and Sanger sequencing before being subjected to dual-platform sequencing on the Illumina NovaSeq 6000 and the Oxford Nanopore MinION Mk1B, generating 4.5 and 6.3 gigabytes of data respectively.

The assembly pipeline was carefully constructed to leverage the strengths of both sequencing platforms. Long Nanopore reads were error-corrected with NECAT and assembled into contigs using Canu v2.2, followed by scaffolding with ntLink-Longstitch and gap filling with the Samba module from MaSuRCA. Five rounds of polishing with Racon using Nanopore reads and four rounds of POLCA using Illumina reads refined the base-level accuracy. The result was a draft genome of 36.88 megabases distributed across 415 scaffolds with an N50 of 301.91 kilobases and a GC content of 43.44 percent. This assembly is larger than the previously reported SOLiD-based Ac2VRR assembly at 34.56 megabases and slightly smaller than the PacBio-based Ac2VPB assembly at 38.96 megabases, positioning the Indian isolate within the expected genomic range for race 2.

Quality assessment using the BUSCO pipeline with the stramenopiles_odb10 dataset confirmed exceptional completeness, with 97 percent complete, 1 percent fragmented, and 2 percent missing single-copy orthologs when using Augustus-predicted gene models, and a perfect 100 percent recovery with the MetaEuk pipeline. Gene prediction using BRAKER2, which integrates RNA-seq transcript evidence from four publicly available datasets with protein homology from 31 reference Oomycota proteomes, identified 13,715 protein-coding genes distributed at an average of 359.03 genes per megabase. Of these, 11,556 genes were functionally annotated using eggNOG-mapper, InterProScan, and databases including GO, KEGG, COG, Pfam, and CAZy, representing 84.2 percent of the predicted gene complement.

A striking feature of the genome is its repetitive landscape. Transposable elements and other repeat sequences occupy 24.29 percent of the assembly, with long terminal repeat retroelements, particularly the Copia/Ty1 family at 5.6 percent, being the most abundant class, followed by long interspersed nuclear elements at 7.3 percent. The researchers also identified 1,039 simple sequence repeats with a relative abundance of 28.16 SSRs per megabase, dominated by mononucleotide motifs, particularly A/T repeats at 77.7 percent among mononucleotides. These microsatellite markers provide a valuable resource for future population genetic studies and molecular marker development aimed at tracking pathogen diversity across geographic regions.

Perhaps the most biologically significant contribution of this study lies in the characterization of the pathogen’s secretome and effector repertoire. Using SignalP v6.0, the team predicted 688 secretory proteins with N-terminal signal peptides, which were then filtered through TMHMM to remove those with transmembrane domains, yielding 526 candidate secretory proteins. EffectorP 3.0 subsequently classified 355 of these as putative effectors, with 304 categorized as cytoplasmic and 51 as apoplastic. Among the annotated effectors, 141 had recognizable homologs while 214 were entirely novel, underscoring the evolutionary divergence of A. candida from other well-characterized oomycetes such as Phytophthora and Hyaloperonospora.

The effector classification revealed several conserved families with important roles in pathogenesis. Six crinkling and necrosis (CRN) class effectors were identified, though only one carried a signal peptide, suggesting that the remaining five may function intracellularly or through non-canonical secretion mechanisms. Six elicitins, a highly conserved family of secreted proteins known to trigger defense responses in plants, were annotated, along with three cellulose-binding elicitor lectins (CBELs) containing cellulose-binding and Apple domains. Notably, the search for classical RXLR motifs yielded no hits, consistent with previous reports that A. candida lacks the canonical RXLR translocation motif found in many oomycete effectors. Instead, the researchers identified 13 gene models carrying a variant Ac-RxL motif, R(SQK)L(FRKE), near the N-terminus, and 15 CCG-type effectors defined by a redefined consensus motif of CxxCxxxxxG.

To validate the biological relevance of these predicted effectors, the team performed quantitative reverse-transcription PCR on infected B. juncea plants at 1, 7, and 12 days post-inoculation. Of the 43 secretory protein genes selected for expression analysis, 26 showed significant differential expression, with most being strongly induced during the later stages of infection. Genes encoding CRN effectors, elicitins, CBELs, CCG-type effectors, and RxL-type effectors all showed elevated expression at 7 and 12 days post-inoculation, with individual genes such as Ac_8954_CRN, Ac_6828_elicitin, and Ac_13392_CCG showing upregulation ranging from 3.02-fold to 12.23-fold compared to the early infection timepoint. This temporal pattern suggests a coordinated deployment of effectors during the transition from initial colonization to sustained biotrophic growth within the host.

Phylogenomic analysis using average nucleotide identity (ANI) revealed that the Indian Ac2V Delhi isolate shares 99.64 percent ANI with the Canadian Ac2VPB assembly and 99.53 percent with Ac2VRR, confirming that all three belong to the same race 2 lineage despite their geographic separation. However, the identification of 62 novel contigs totaling 234,330 base pairs, representing 0.63 percent of the genome, that showed no sequence similarity to the Canadian isolates provides evidence of strain-specific genomic variation. These unique contigs were enriched for long terminal repeat retrotransposons and mobile genetic element-associated proteins, suggesting that transposable element activity may be a significant driver of structural variation within the race. The availability of this high-quality genome assembly, deposited under NCBI accession JBBMRL000000000, will serve as a foundational resource for comparative genomics, effector discovery, and the development of durable disease management strategies for white rust in Indian mustard and other brassica crops globally.

Subject of Research: Whole genome sequencing and secretome analysis of the white rust pathogen Albugo candida infecting Brassica juncea

Article Title: Comprehensive whole genome hybrid assembly unravels genes and secretome of Albugo candida (Indian isolate), causing white rust disease in Brassica juncea

Article References: Mehta, S., Awasthi, L., Tomar, R., Yadava, Y. K., Rao, M., Iyyappan, Y., Bashyal, B. M., Akhtar, J., Bhattacharya, R. C., Rai, P. K., & Gupta, A. K. (2026). Comprehensive whole genome hybrid assembly unravels genes and secretome of Albugo candida (Indian isolate), causing white rust disease in Brassica juncea. Frontiers in Fungal Biology, 7, Article 1756853. https://doi.org/10.3389/ffunb.2026.1756853

Image Credits: AI Generated

DOI: 10.3389/ffunb.2026.1756853

Keywords: Albugo candida, Brassica juncea, white rust, genome assembly, effectors, secretome, oomycete, Nanopore sequencing, Illumina, transposable elements, plant pathology, biotrophic pathogen