Fusarium wilt of watermelon is a devastating vascular disease. The fungus enters through the roots, threads its way into the water-conducting xylem vessels, and effectively strangles the plant from within, disrupting water transport until leaves wilt and the plant dies. What makes the disease especially difficult to control is the pathogen’s biology: the fungus can survive in soil for many years, new races capable of overcoming resistant cultivars continue to emerge, and effective field treatments remain scarce. Because Fusarium oxysporum as a species can infect more than 150 plants, including cotton, tomato, melon, and banana, understanding how its virulence works has implications far beyond the watermelon patch.

The Zhejiang team, led by Fengming Song, focused on a class of molecules known as effector proteins. Fungal pathogens secrete these proteins in large numbers during infection to manipulate or suppress plant immunity. Effectors are broadly divided into two categories based on where they operate: apoplastic effectors, which act in the space outside plant cell membranes, and cytoplasmic effectors, which function inside host cells. In the tomato-infecting form of the fungus, researchers previously identified a family of Secreted In Xylem, or SIX, proteins, many of which proved pivotal to disease. But for the watermelon-infecting strain, the functional repertoire of effectors remained largely unexplored.

The protein at the center of the new study, FonCHRD, caught the researchers’ attention because it carries a chordin, or CHRD, domain whose function in fungi was unknown. Chordin is famous in developmental biology: it was first isolated from the Spemann organizer of the frog embryo Xenopus laevis, where it antagonizes bone morphogenetic proteins and helps pattern the body axis. CHRD domain proteins also appear in green algae, where they are linked to stress responses, and have been implicated as potential biomarkers in human cancers. In the tomato wilt fungus, a CHRD-containing protein had been flagged as a likely pathogenicity effector, but nobody had experimentally pinned down what such a protein actually does in a plant-pathogenic fungus.

FonCHRD turned out to be a compact protein of 193 amino acids with a 16-amino-acid signal peptide at its N-terminus, the molecular postal address that directs proteins into the conventional secretion pathway. The team confirmed this experimentally using a yeast secretion trap assay: yeast cells engineered to carry the FonCHRD signal peptide could grow on selective medium and reduce a colorless compound to a red precipitate, proving the peptide genuinely drives secretion. Bioinformatic tools classified the protein as an apoplastic or cytoplasmic effector, and structural modeling showed its three-dimensional fold closely resembles known CHRD domain proteins. Notably, CHRD-containing proteins also occur in other major plant pathogens, including the rice blast fungus Magnaporthe oryzae, Botrytis cinerea, and Verticillium dahliae, suggesting an evolutionarily conserved role.

Expression profiling added a telling clue. FonCHRD was most active in macroconidia, the banana-shaped spores that spread the fungus, and its expression surged roughly thirtyfold when spores were exposed to watermelon root tissue, peaking at 36 hours. That pattern, a gene quiet in resting mycelium but switched on by host contact, is a hallmark of effectors deployed at the earliest stages of infection. To test function, the researchers deleted the gene using targeted gene replacement and then restored it in a complemented strain. The deletion mutant, designated ΔFonCHRD, grew more slowly on culture medium, with colony diameters reduced by 44 percent on potato dextrose agar and 26 percent on minimal medium. It also produced about a third fewer macroconidia, and those spores were visibly abnormal: curled, shorter, and carrying fewer cross-walls, or septa, than wild-type spores. Intriguingly, spore germination was unaffected, and the mutant showed no heightened sensitivity to cell wall, oxidative, or salt stress, indicating the protein’s role is selective rather than a general stress function.

The pathogenicity results were dramatic. Twenty-one days after inoculation, 71 percent of watermelon plants infected with the wild-type fungus had severely wilted or died, while only 7 percent of plants exposed to the deletion mutant had died, translating to a 51 percent drop in disease severity. Plants inoculated with the mutant also began dying three to six days later than those facing the wild type, and at 27 days, all wild-type-infected plants were dead while only 43 percent of mutant-infected plants had succumbed. Reintroducing the gene restored full virulence, confirming the effect was due to FonCHRD itself. Quantitative PCR revealed that fungal biomass inside infected roots was reduced by 56 percent and inside stems by a striking 85 percent, yet the mutant penetrated cellophane membranes as efficiently as the wild type. The picture that emerges is that FonCHRD does not help the fungus break into the plant; it helps the fungus spread once inside, particularly as it moves from root to stem through the vascular system.

To see where the protein acts in plant cells, the team tagged FonCHRD with green fluorescent protein and expressed it transiently in leaves of Nicotiana benthamiana, a widely used experimental plant. The protein appeared in the cytoplasm, nucleus, and plasma membrane, and formed punctate spots whose significance remains unclear. When the researchers induced plasmolysis with mannitol to shrink the cell membrane away from the wall, fluorescent signal accumulated in the apoplastic space, confirming that FonCHRD is secreted outside plant cells and that its signal peptide directs this targeting. A variant lacking the signal peptide failed to reach the apoplast, while a hybrid variant in which the native peptide was replaced with a plant apoplastic targeting sequence restored apoplastic localization.

The most striking results came from immune suppression assays. INF1, an elicitor from the potato late blight pathogen, and BAX, a pro-apoptotic mouse protein, both reliably trigger programmed cell death in Nicotiana leaves, a defense reaction analogous to the hypersensitive response plants use to wall off invaders. When FonCHRD was co-expressed with either inducer, the cell death response disappeared or was markedly alleviated. The protein also dampened the expression of six defense-related genes, including pathogenesis-related genes NbPR1 and NbPR2, that INF1 and BAX normally switch on. Remarkably, the signal peptide was dispensable for this suppression: even the variant lacking it blocked cell death, implying that FonCHRD acts inside plant cells to interfere with immunity, while the peptide serves mainly to route the protein to the apoplast. This dual behavior mirrors other Fusarium effectors such as FoSSP17 and FoCupin1, which also suppress cell death independently of their signal peptides.

What makes FonCHRD unusual among Fusarium effectors is that it is not merely a virulence accessory. Most known effectors, such as FolAsp, FoRnt2, and FoSSP17, are dispensable for the fungus’s basic biology and live on mobile pathogenicity chromosomes. FonCHRD, by contrast, is woven into vegetative growth, asexual reproduction, and spore morphology, making it a pleiotropic factor whose loss cripples the organism in multiple ways at once. That combination of housekeeping and warfare roles suggests the sole CHRD domain protein in the fungal genome may have been recruited from an ancestral developmental function into pathogenesis, echoing chordin’s deep role in animal development. The authors caution that the reduced virulence of the mutant could partly reflect its impaired growth, and that the protein’s precise biochemical activity and its interaction partners in both fungus and host remain to be identified. Still, the study delivers a clear strategic insight: a pathogen that must both build its own body and dismantle the plant’s defenses depends on a single secreted protein to do both. Targeting such dual-purpose molecules, whether through resistance breeding, engineered decoys, or small molecules that block their secretion or folding, could offer a way to blunt Fusarium wilt that the fungus would find hard to evade, since losing the protein costs it far more than a single weapon.

Subject of Research: Functional characterization of the secreted effector protein FonCHRD in the watermelon Fusarium wilt fungus Fusarium oxysporum f. sp. niveum

Article Title: The secreted protein FonCHRD is essential for vegetative growth, asexual reproduction, and pathogenicity in watermelon Fusarium wilt fungus

Article References: Lou, J., Wang, J., Zeng, S., Xiong, X., Guo, M., Li, D., & Song, F. (2024). The secreted protein FonCHRD is essential for vegetative growth, asexual reproduction, and pathogenicity in watermelon Fusarium wilt fungus. Crop Health, 2(1), Article 16. https://doi.org/10.1007/s44297-024-00036-x

Image Credits: AI Generated

DOI: 10.1007/s44297-024-00036-x

Keywords: FonCHRD, Fusarium oxysporum f. sp. niveum, watermelon Fusarium wilt, effector protein, plant immunity, chordin domain, pathogenicity, programmed cell death, apoplast, macroconidia, vascular wilt, secreted protein