Stripe rust is a formidable adversary. Under favorable environmental conditions, the disease can strip wheat fields of up to 60 percent of their yield, and the pathogen’s highly dynamic populations continually evolve new races that overcome resistant cultivars. Because the fungus is an obligate biotroph, meaning it must feed on living host tissue, it has evolved sophisticated strategies to suppress plant defenses while quietly extracting nutrients. Farmers currently rely on a combination of fungicides and resistance genes, but the constant arms race between wheat and its attacker makes it essential to identify new layers of immune regulation that could be exploited for crop protection. The new study suggests that gas signaling, long overlooked in this context, may represent exactly such a layer.

The team began by testing whether exogenous hydrogen sulfide could influence disease outcomes directly. Wheat seedlings inoculated with the virulent Pst race V26 were treated with the hydrogen sulfide donor sodium hydrosulfide, or NaHS, at concentrations of 100, 300, and 500 micromolar. Compared with untreated inoculated plants, the treated seedlings showed markedly attenuated disease symptoms and reduced fungal proliferation, with the strongest protective effect at the highest dose. A slow-releasing hydrogen sulfide donor called GYY4137 produced similar suppression of pathogen development, while a chemical scavenger that removes hydrogen sulfide increased necrotic lesion area in an otherwise resistant interaction. Quantitative microscopy revealed that the gas treatment boosted the accumulation of reactive oxygen species and hypersensitive cell death at infection sites in a concentration-dependent manner, while fungal hyphal area and length correspondingly shrank. The effect was unambiguous: hydrogen sulfide enhances wheat immunity in a dose-dependent fashion.

To understand how the gas exerts this protection, the researchers turned to the molecular mechanism most commonly associated with hydrogen sulfide signaling: protein persulfidation. This post-translational modification converts the thiol groups of cysteine residues into persulfides, altering protein activity, interactions, and stability in a rapid and reversible way. Using comparative persulfidation proteomics, the team catalogued the persulfidated proteins in wheat leaves infected with Pst races CYR32 and V26 and compared them with uninfected controls. The scale of the modification was striking: 16,473 persulfidated proteins were identified in total, of which 1,053 were uniquely modified in infected samples. Gene ontology analysis showed that the proteins specifically persulfidated upon infection were enriched in biological processes tied to immunity and stress adaptation, including responses to other organisms, stress responses, and signal transduction.

Within the stress-response category, one protein stood out. TaATG6c, a wheat homolog of the conserved autophagy regulator ATG6/Beclin1, was the only protein with a direct functional association with the autophagy pathway. Autophagy is the cell’s recycling system, in which double-membraned vesicles called autophagosomes engulf damaged organelles, protein aggregates, and even invading pathogens for degradation. In plants, this process is intimately connected to immunity: it restricts pathogen invasion, modulates immune signaling, regulates programmed cell death, and maintains reactive oxygen species homeostasis. ATG6 serves as a central scaffold within the class III phosphatidylinositol-3-kinase complex that initiates autophagosome formation, making it a plausible point of control for a gas signal that needs to act quickly during infection.

Site-specific mass spectrometry pinpointed exactly where hydrogen sulfide attaches to TaATG6c. Two cysteine residues, Cys177 and Cys180, each displayed the characteristic mass shift of 31.97207 daltons that signals persulfidation. A modified biotin-switch assay confirmed the modification: NaHS treatment induced a concentration-dependent increase in TaATG6c persulfidation, the reducing agent DTT abolished the signal, and substituting the two cysteines with alanines completely eliminated the NaHS-induced modification. Notably, the two sister paralogs TaATG6a and TaATG6b showed no detectable persulfidation, underscoring the specificity of TaATG6c as the hydrogen sulfide-responsive member of the family. Structural modeling based on AlphaFold added a mechanistic dimension: both modified residues sit within the central coiled-coil domain, exposed on the protein surface at the interface where TaATG6c binds its partner ATG14. The modeling predicted that mutating these residues weakens the TaATG6c–ATG14 interaction by reducing hydrogen bonds at the interface, suggesting that persulfidation fine-tunes the assembly of the autophagy initiation complex without relocating the protein within the cell.

Functional experiments then established that TaATG6c is a genuine positive regulator of stripe rust resistance. Using virus-induced gene silencing targeting a conserved region shared by the three TaATG6 paralogs, the researchers suppressed TaATG6 expression by roughly 70 percent. When these silenced plants were challenged with the avirulent race CYR23, they developed more severe disease symptoms and carried significantly higher fungal biomass than controls. Microscopy revealed enlarged infection sites with increased hyphal growth. Intriguingly, the silenced plants accumulated more reactive oxygen species and more cell death, yet these responses failed to contain the fungus. The authors interpret this as evidence that excessive ROS arising from impaired autophagy is not sufficient to restrict pathogen proliferation, because autophagy normally helps maintain oxidative balance. Supporting this view, the autophagy activator lithium chloride suppressed Pst growth, while the inhibitor 3-methyladenine worsened necrotic lesions. TaATG6 expression itself was strongly induced during infection, peaking at 48 hours post-inoculation and reaching higher levels in the incompatible interaction than in the compatible one.

The decisive experiment connected all the threads. In control plants, NaHS treatment markedly alleviated disease symptoms caused by the compatible race CYR32, but this protective effect was substantially attenuated in TaATG6-silenced plants, demonstrating that the gas requires TaATG6c to deliver its full benefit. Conversely, transient overexpression of TaATG6 enhanced resistance, whereas overexpression of the C177A/C180A mutant failed to do so, proving that the two cysteine residues are critical for the protein’s resistance-promoting function. Endogenous biotin-switch assays showed that TaATG6c persulfidation follows a dynamic, pathogen-responsive pattern: it rises rapidly after infection, peaks at 24 hours, and gradually declines thereafter, while the cysteine mutant remained flat throughout. Finally, the team tracked the accumulation of lipidated ATG8, a hallmark of autophagosome formation. Both hydrogen sulfide treatment and Pst infection increased ATG8–PE levels in wild-type plants, and the combined treatment amplified the effect, but in TaATG6-silenced plants the basal level of ATG8–PE dropped and neither stimulus could induce it.

Taken together, the results sketch a coherent model of a redox-regulated immune circuit. When the stripe rust fungus attacks, hydrogen sulfide signaling surges, and the gas chemically tags TaATG6c at Cys177 and Cys180. This persulfidation strengthens the protein’s engagement with the autophagy initiation machinery, promoting autophagosome formation at precisely the moment the plant needs to deploy cellular recycling against the invader. Because the modification is rapid and reversible, it allows wheat to coordinate its defense within a precise temporal window rather than simply cranking up protein abundance. The work also resonates with earlier findings in Arabidopsis, where persulfidation of the autophagy protein ATG18a enhances autophagosome formation under stress, suggesting that sulfur-based control of autophagy may be a conserved feature of plant biology. For breeders and crop scientists, the study points to TaATG6c and its persulfidation sites as potential molecular targets for engineering or selecting wheat varieties with stronger, more durable resistance to stripe rust, and it elevates a humble, foul-smelling gas to the status of a key player in the global fight for food security.

Subject of Research: Hydrogen sulfide signaling and autophagy-mediated immunity in wheat against stripe rust

Article Title: Hydrogen sulfide promotes wheat immunity against stripe rust through TaATG6c persulfidation

Article References: Hydrogen sulfide promotes wheat immunity against stripe rust through TaATG6c persulfidation. (n.d.). https://doi.org/10.1007/s44154-026-00292-7

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00292-7

Keywords: hydrogen sulfide, wheat, stripe rust, Puccinia striiformis, autophagy, TaATG6c, persulfidation, plant immunity, ATG8, redox signaling, crop disease resistance, post-translational modification

The research, led by Ying Zhou, Xue Zhang, Tiancong Qi, and Zi-Han Wang working with senior authors Lu-Jun Yu and Qin-Fang Chen at Sun Yat-Sen University and their collaborators, began with a deliberately simple strategy. The team used the central autophagy protein ATG8e as bait in a yeast two-hybrid screen, fishing an Arabidopsis cDNA library for any protein capable of physically binding to it. ATG8 sits at the heart of autophagosome formation, the process by which a double-membrane vesicle engulfs cellular cargo before delivering it to the vacuole for destruction. Proteins that dock onto ATG8 typically do so through a short peptide signature known as the ATG8-interacting motif, or AIM, defined by the consensus sequence of an aromatic residue followed by two variable positions and a bulky hydrophobic one. Among the fifteen candidate interactors that emerged from the screen, one stood out: MEMBRANE ATTACK COMPLEX/PERFORIN-LIKE 2, or MACP2, a member of a protein family better known for its role in immunity and programmed cell death.

The MACPF family is famous in animal biology. Members of this family, including the complement membrane attack complex and perforin, oligomerize into ring-shaped pores that punch holes in target membranes, a mechanism central to immune defense and development. Plants encode a small set of MACPF proteins, and in Arabidopsis four of them carry the conserved pore-forming domain: CAD1, NSL1, MACP1, and MACP2. Previous work had shown that MACP2 regulates programmed cell death and influences resistance to bacterial pathogens and susceptibility to necrotrophic fungi, and its transcript levels rise in response to a range of biotic and abiotic stresses. But its involvement in autophagy had never been suspected. The new study confirmed the physical interaction between MACP2 and ATG8e using two independent methods: co-immunoprecipitation in Arabidopsis mesophyll protoplasts, where tagged ATG8e pulled down tagged MACP2 from cell extracts, and bimolecular fluorescence complementation, in which the two proteins reconstituted a fluorescent signal at the plasma membrane only when co-expressed. Crucially, when the researchers mutated two conserved residues within the AIM, changing phenylalanine 134 and leucine 137 to alanines, the interaction vanished entirely, proving that MACP2 docks onto ATG8 through the canonical autophagy receptor motif.

With the interaction established, the team turned to genetics to ask what MACP2 actually does in a living plant. They compared wild-type Arabidopsis with macp2 knockout mutants and with lines overexpressing the protein, subjecting all genotypes to carbon starvation by transferring them to continuous darkness and to nitrogen starvation by growing them on nitrogen-free medium. The results were striking and, at first glance, counterintuitive. Plants lacking MACP2 were more tolerant of starvation, retaining greener leaves and higher chlorophyll content than wild type, while plants overproducing MACP2 senesced prematurely and wilted faster under the same conditions. Under normal growth the differences were subtle, appearing only as the plants aged, with overexpression lines yellowing by five to six weeks while the knockouts stayed green longer than their wild-type counterparts. This pattern, in which too much of a protein mimics the hypersensitivity of autophagy-defective mutants, suggested that MACP2 acts as a positive regulator of autophagy induction: without it, the starvation signal is blunted, and with too much of it, the signal becomes destructive.

The signal in question, the study reveals, is hydrogen peroxide. Reactive oxygen species have long been recognized as double-edged messengers in plant cells: at low concentrations they act as second messengers that coordinate growth and stress responses, but in excess they oxidize proteins, lipids, and DNA beyond repair. Outside the cell, in the apoplast, hydrogen peroxide is generated primarily by plasma membrane NADPH oxidases of the RBOH family, particularly RBOHD and RBOHF. Earlier work had hinted that these apoplastic reactive oxygen species are required for autophagosome formation during hypoxia, but the molecular channel connecting extracellular peroxide to the intracellular autophagy machinery had never been identified. Using an Amplex Red fluorescence assay, the researchers measured total hydrogen peroxide levels across their genotypes and found that MACP2-overexpressing plants accumulated significantly more peroxide during senescence and starvation, while macp2 knockouts accumulated significantly less. To visualize peroxide influx directly, they deployed HyPer, a genetically encoded fluorescent biosensor specific for hydrogen peroxide, and showed that the peroxide-dependent fluorescence ratio in root cells rose sharply after carbon starvation in wild-type seedlings but remained substantially lower in macp2 mutants both before and after starvation.

The picture that emerges is one of MACP2 acting as a gated doorway for stress chemistry. The authors propose that in response to senescence or nutrient deprivation, MACP2 proteins assemble into homo- or hetero-polymeric pores at the plasma membrane, structurally analogous to the membrane attack complexes of animal immunity, and that these pores allow extracellular hydrogen peroxide, produced by RBOH oxidases, to flood into the cytoplasm. There, the peroxide functions as a signal that triggers the initiation of autophagosome biogenesis. Supporting this model, supplying the antioxidant glutathione to starving seedlings completely rescued the starvation hypersensitivity of MACP2-overexpressing lines and partially rescued the atg5-1 autophagy mutant, demonstrating that the damaging phenotypes are chemically driven by peroxide rather than by some unrelated consequence of MACP2 abundance. The salicylic acid pathway was also implicated: overexpression lines accumulated elevated levels of salicylic acid and its glycoside SAG, showed upregulation of senescence-associated and defense genes including SAG13, SAG29, PR1, and WRKY53, and lost their accelerated senescence when crossed into eds1 or pad4 salicylic acid signaling mutants. Salicylic acid and reactive oxygen species, the authors note, appear to form a mutually reinforcing amplification loop that drives both autophagy induction and senescence.

Perhaps the most elegant evidence for MACP2’s role came from epistasis experiments with the autophagy mutant atg5-1. The atg5-1 mutant, defective in a core autophagy conjugation factor, displays the classic autophagy-defective phenotype: early leaf yellowing, runaway peroxide accumulation, and extreme sensitivity to carbon and nitrogen starvation. Remarkably, when the researchers crossed macp2 into the atg5-1 background, the double mutants were substantially rescued, showing delayed senescence, higher chlorophyll content, greater starvation tolerance, and dramatically lower hydrogen peroxide levels than atg5-1 alone. In other words, removing the peroxide doorway suppressed the damage caused by removing the recycling machinery itself, placing MACP2 genetically upstream of ATG5 as a regulator of the peroxide signal that both induces autophagy and, when unchecked, kills the cell. Consistent with this, direct measurements of autophagosome formation using an eGFP-ATG8e reporter showed that macp2 mutants formed fewer starvation-induced autophagic puncta and released less free eGFP, a readout of autophagic flux, while overexpression lines formed more. Treating seedlings with exogenous hydrogen peroxide or the salicylic acid agonist BTH likewise induced fewer puncta in macp2 and more in overexpressors, confirming that MACP2 channels the peroxide signal into autophagosome biogenesis.

The story acquires a final twist in the regulation of MACP2 itself. Under normal conditions, a MACP2-YFP fusion localizes to the plasma membrane, but after twelve hours of carbon or nitrogen starvation the fluorescent signal redistributes into cytoplasmic vesicles that colocalize with the autophagosome marker mCherry-ATG8f. Prolonged darkness causes MACP2 protein abundance to collapse after roughly 24 to 48 hours, and this degradation is blocked by concanamycin A, an inhibitor of vacuolar degradation, but not by the proteasome inhibitor MG132, indicating that MACP2 is dismantled by autophagy rather than by the 26S proteasome. Protein stability assays in protoplasts from atg5-1 and rpn10-1 mutants, the latter lacking a dual ATG8 and ubiquitin receptor, confirmed that MACP2 degradation requires the autophagy machinery and likely involves RPN10 acting as a selective autophagy receptor. The researchers also found that MACP2 lacking a functional AIM degrades more slowly and that its stability no longer depends on ATG8e, tying the feedback degradation directly to the ATG8 interaction. This creates a self-limiting circuit: early in starvation, MACP2 pores admit peroxide, peroxide induces autophagy, and autophagy preserves cellular homeostasis; but as peroxide continues to accumulate, MACP2 itself is loaded onto autophagosomes and destroyed, throttling the peroxide influx and protecting the cell from self-destruction during extended deprivation.

The implications of this work extend well beyond a single weed species. It identifies, for the first time, a molecular conduit through which extracellular reactive oxygen species gain access to the autophagy induction machinery in plants, and it adds MACP2 to a growing list of membrane proteins, including the aquaporins PIP1;4 and PIP2;1, that govern hydrogen peroxide trafficking across plant membranes. It also resonates with recent findings in animal cells, where the pore-forming protein gasdermin D mediates the release of mitochondrial reactive oxygen species, suggesting that pore-mediated peroxide transport may be a broadly conserved principle of cellular stress signaling. For agriculture, the prospect of tuning MACP2 or its relatives to adjust the sensitivity of crops to nutrient stress, darkness, or pathogen attack is an obvious long-term goal, though the authors caution that the precise architecture of the proposed MACP2 pore and the exact mechanism by which RPN10 targets MACP2 for degradation remain to be worked out. What is already clear is that the plant’s decision to consume itself in hard times is not a spontaneous collapse but a signal-driven choice, and that the gatekeeper of that signal is a protein borrowed from the ancient arsenal of membrane attack.

Subject of Research: Role of the Arabidopsis pore-forming protein MACP2 in linking extracellular reactive oxygen species to autophagy induction during nutrient starvation

Article Title: Arabidopsis MACP2 contributes to autophagy induction by modulating starvation-induced reactive oxygen species homeostasis

Article References: Zhou, Y., Zhang, X., Qi, T., Wang, Z.-H., Wang, Y., Wang, L.-N., Zeng, Y.-L., He, H., Jiang, L., Xie, D., Xiao, S., Yu, L.-J., & Chen, Q.-F. (2025). Arabidopsis MACP2 contributes to autophagy induction by modulating starvation-induced reactive oxygen species homeostasis. Advanced Biotechnology, 3(3), Article 25. https://doi.org/10.1007/s44307-025-00078-4

Image Credits: AI Generated

DOI: 10.1007/s44307-025-00078-4

Keywords: autophagy, MACP2, Arabidopsis, reactive oxygen species, hydrogen peroxide, ATG8, nutrient starvation, leaf senescence, membrane attack complex, salicylic acid, plant stress, RPN10