Anthracnose, driven by fungal species in the genus Colletotrichum, is a persistent problem for sweet orange growers. The disease scars leaves and fruit, depresses yields, and degrades the commercial quality of harvests, and conventional management relies heavily on fungicides whose repeated use raises environmental and resistance concerns. Bacillus subtilis has long been recognized as a biocontrol agent capable of suppressing pathogens directly and of inducing host defense responses, but the host-side transcriptional mechanisms that accompany its protective effect in sweet orange remained poorly defined. The new study set out to close that gap by asking which members of the WRKY transcription factor family respond when the pathogen attacks and how those responses change when B. subtilis is applied as an intervention.

WRKY transcription factors are one of the largest families of plant-specific regulators, named for a highly conserved amino acid motif in their DNA-binding domain, and they are famous for orchestrating responses to stress, including pathogen attack. They bind promoter sequences known as W-boxes, which are enriched in the regulatory regions of many defense-related genes. To map this regulatory landscape in sweet orange, the researchers systematically searched the reference genome DVS_A1.0 and identified 55 CsWRKY family members. They then characterized the structural features of these genes and analyzed how their expression changed across an experimental system that compared healthy controls, plants infected with the anthracnose pathogen, and infected plants that also received B. subtilis treatment.

The phenotypic results were striking in their clarity. When the researchers measured lesion diameters on leaves, the infected plants that had not received the bacterium, designated the BT group, developed lesions averaging 8.72 plus or minus 0.84 millimeters. In contrast, plants in the BST group, which were infected and then treated with B. subtilis, showed lesions averaging 5.47 plus or minus 0.66 millimeters. That difference corresponds to a 37.3 percent reduction in lesion size attributable to the bacterial intervention, a magnitude of protection that is biologically meaningful for a disease that otherwise spreads aggressively through orchards.

Behind that visible symptom reduction lay an extensive transcriptional reprogramming event. Transcriptomic analysis revealed that 28 CsWRKY genes were differentially expressed when infected plants were compared with healthy controls, and 27 were differentially expressed when B. subtilis-treated infected plants were compared with infected plants alone. This near-overlapping set of responsive genes indicates that the WRKY family is broadly engaged during anthracnose infection and that the biocontrol bacterium substantially reshapes that engagement rather than acting on a single isolated pathway. The scale of the response suggests that B. subtilis does not simply kill the pathogen at the leaf surface but actively reorganizes the host’s internal defense signaling network.

To move from a list of responsive genes to a candidate regulator, the team integrated multiple lines of evidence. They examined expression patterns across treatments, constructed a predicted protein-protein interaction network to see which WRKY proteins occupied central positions in the inferred regulatory web, and validated key expression changes using reverse transcription quantitative PCR. This convergence of approaches prioritized CsWRKY40 as a candidate regulatory node. Its expression was downregulated under infection alone but restored and upregulated when B. subtilis was applied, a pattern consistent with a gene whose activity supports the protective state induced by the beneficial bacterium.

Beyond its expression dynamics, CsWRKY40 carries molecular features that hint at how its function might be controlled in the cell. The researchers used the NetPhos 3.1 tool to predict phosphorylation sites across the protein and found multiple potential sites, suggesting that CsWRKY40 could be regulated post-translationally through phosphorylation, a common mechanism by which plant cells rapidly modulate transcription factor activity during immune signaling. While the study stops short of functional validation, the presence of these predicted sites provides a concrete starting point for future experiments aimed at determining how the protein’s activity is switched on or off during the interaction between the pathogen, the bacterium, and the host.

Perhaps the most ambitious component of the study was its genome-wide promoter scan. The researchers searched the entire sweet orange genome for genes containing W-box motifs in their promoter regions and cross-referenced those candidates with the differential expression data from the BT versus CK and BST versus BT comparisons. This analysis identified 5,143 W-box-containing candidate genes that were responsive in at least one of the treatment contrasts. The team then integrated Gene Ontology, KEGG and KO orthology, NR protein database, Swiss-Prot, and protein-domain annotations to classify these candidates functionally. Of the total, 1,736 were classified as defense-associated candidates, falling into categories that include reactive oxygen species and redox homeostasis, general defense responses, hormone signaling, MAPK signaling cascades, cell wall remodeling, secondary metabolism, and pathogenesis-related functions.

That functional breadth is significant because it maps the potential downstream territory that CsWRKY40 and other responsive WRKY factors might govern. Reactive oxygen species bursts are among the earliest and most consequential events in plant immune signaling, and maintaining redox balance determines whether that burst is protective or damaging to host tissue. Hormone pathways, particularly those involving salicylic acid and jasmonic acid, cross-talk extensively to shape defense outcomes against different classes of pathogens. MAPK cascades relay signals from pathogen perception to transcriptional responses, and cell wall remodeling determines the physical barriers a pathogen must breach. Secondary metabolism supplies antimicrobial compounds, and pathogenesis-related proteins are canonical markers of induced defense. The fact that so many W-box-containing genes across these categories shifted expression in response to infection and to B. subtilis treatment suggests that the bacterium’s protective effect operates through a coordinated rewiring of multiple defense modules rather than a single linear pathway.

The authors are careful to frame CsWRKY40 as a candidate rather than a confirmed regulator, and that distinction matters. Correlation between expression patterns and phenotypic outcomes, even when supported by network predictions and qPCR validation, does not establish causation. Functional validation, whether through gene knockout, overexpression, or complementation in stable transgenic lines, will be needed to determine whether CsWRKY40 genuinely drives the protective response or simply correlates with it. Nevertheless, the study provides a well-defined starting point for such experiments and contributes a curated resource of 55 CsWRKY family members, their structural features, and their expression responses to both pathogen challenge and biocontrol intervention. For citrus growers and plant pathologists, the work adds molecular texture to a strategy that has long been used empirically in the field, and it points toward a future where beneficial bacteria might be deployed not just as living pesticides but as precision tools that engage specific host regulatory circuits to strengthen crop immunity from within.

Subject of Research: Identification of the WRKY transcription factor CsWRKY40 as a candidate regulator of Bacillus subtilis-mediated anthracnose suppression in sweet orange

Article Title: A genome-wide and transcriptome-integrated identification of CsWRKY40 as a candidate regulator associated with B. subtilis-mediated reduction of anthracnose symptoms in sweet orange

Article References: Wang, H., Ding, S., Wang, J., Pan, W., Wan, N., Sai, J., Li, Y., Zhang, L., Yao, W., Xu, R., Zhang, H., & Liu, X. (2026). A genome-wide and transcriptome-integrated identification of CsWRKY40 as a candidate regulator associated with B. subtilis-mediated reduction of anthracnose symptoms in sweet orange. BMC Plant Biology. https://doi.org/10.1186/s12870-026-10069-9

Image Credits: AI Generated

DOI: 10.1186/s12870-026-10069-9

Keywords: sweet orange, Citrus sinensis, anthracnose, Colletotrichum, Bacillus subtilis, WRKY transcription factors, CsWRKY40, biocontrol, transcriptomics, plant immunity, W-box, gene regulation