ZmGDIa-hel counters the RBSDV-induced reduction of active gibberellins to alleviate maize rough dwarf virus disease

Plant viruses transmitted by vectors cause major crop losses. Plants deploy active and recessive resistance; many recessive resistance genes involve translation initiation factors, but other loss-of-susceptibility genes include ZmGDIa-hel. MRDD, caused by fijiviruses such as RBSDV, produces dwarfism, shortened internodes, abnormal reproductive structures, dark-green leaves, and enations. RBSDV encodes P7-1, which forms tubules and localizes to plasmodesmata for movement. Viral infections often perturb phytohormone balance; prior examples include viral effectors altering auxin, gibberellin, salicylic acid, jasmonic acid, and cytokinin pathways. Gibberellins GA4 and GA1 are key bioactive GAs, inactivated by GA2-oxidases. The authors previously identified a helitron-induced ZmGDIa-hel allele conferring quantitative recessive resistance to MRDD, but the mechanism was unclear. The research questions: how does P7-1 interacting with ZmGDIa initiate MRDD, and how does reduced interaction with ZmGDIa-hel alleviate MRDD? The study identifies ZmGA20x7.3 (a GA2ox) as a key player whose levels and interactions with P7-1 and ZmGDIa/ZmGDIa-hel modulate GA and auxin/cytokinin balance to determine MRDD outcomes.

• Recessive resistance frequently involves eIF4E/eIF4G genes; additional loss-of-susceptibility genes include ZmGDIa-hel, cPGK2, and HuPDIL5-1.
• MRDD has been reported globally; RBSDV is the major East Asian pathogen transmitted by Laodelphax striatellus. RBSDV genome has 10 dsRNA segments; P7-1 (S7-1) forms tubules and uses secretory and actomyosin pathways for movement.
• Viral proteins can reprogram hormone signaling: TMV replicase interacts with Aux/IAA (auxin), RDV P2 reduces GA1 via ent-kaurene oxidase interaction causing dwarfism, rice stripe virus effectors modulate SA/JA crosstalk via OsNPR1, geminivirus AC2/AL2 impacts cytokinin responses, and NLR Sw-5b engages ABA regulators in antiviral defense.
• GAs regulate growth; GA2oxs inactivate GA1/GA4. Overexpression of C19-GA2oxs reduces endogenous bioactive GAs, causing dwarfism. Rice GA2oxs like OsGA2ox3/7 are class I enzymes targeting C19-GAs. These prior findings set the context that viral manipulation of GA metabolism could underlie MRDD symptoms and that GA2oxs are prime candidates in GA inactivation.

• Plant materials: Near-isogenic maize lines NIL-S (ZmGDIa) and NIL-R (ZmGDIa-hel); transgenic maize overexpressing ZmGA20x7.3-GFP and P7-1-GFP in B73; transgenic Arabidopsis overexpressing ZmGA20x7.3 or oligomerization-defective ZmGA20x7.3 variants.
• RBSDV inoculation: Seedlings exposed to viruliferous or non-viruliferous planthoppers for 3 days at emergence; grown in field for phenotyping and sampling at defined dpi.
• Transcriptomics: RNA-seq at 16 dpi comparing NIL-S vs NIL-R; DEGs with fold-change > 2 and P < 0.05; GO/KEGG enrichment; validation by RT-qPCR. Prior Co-IP/MS dataset used to cross-reference candidate interactors.
• Gene/protein interaction assays: Split luciferase complementation (SLC), bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), GST/His pull-downs, and microscale thermophoresis (MST) to quantify binding affinities and map interaction domains between ZmGA20x7.3, ZmGDIa/ZmGDIa-hel, and P7-1.
• Protein engineering: Construction of ZmGA20x7.3 oligomerization-defective mutant (3A) by mutating conserved residues; domain truncations of ZmGDIa/ZmGDIa-hel to identify binding regions.
• Subcellular localization: Transient expression in Nicotiana benthamiana and maize protoplasts with confocal microscopy.
• Oligomerization analyses: Blue Native-PAGE and size-exclusion gel filtration chromatography (Superdex 200 Increase 10/300 GL) with/without GA4; calibration with molecular weight standards; BN-PAGE/SDS-PAGE immunoblotting to validate oligomer sizes and compositions; LC-MS to identify components in multimer peaks.
• Enzyme assays: In vitro GA2ox activity measured by quantifying GA34 production from GA4 (and GA12-based assay framework) across GA4 concentrations; testing effects of adding P7-1, ZmGDIa, or ZmGDIa-hel on ZmGA20x7.3 activity.
• Hormone quantification: UHPLC-MS/MS of GA species (GA1, GA4, GA5, GA9, GA20, GA29, GA34, GA51), IAA (auxin), and cytokinins (iP, iPR, tZ, tZR, cZ, etc.) in upper internodes and upper leaves at 60 dpi.
• Exogenous GA treatments: Spraying GA1, GA3, GA4 on seedlings of B73 and ZmGA20x7.3-OE to assess phenotypic rescue and feedback on ZmGA20x7.3 expression/oligomerization.
• Cytology: TEM/SEM of upper internode tissues to visualize cell wall thickness, membrane integrity, and presence of virions.
• Statistics: Normality tests; Student’s t-test, Mann-Whitney/Wilcoxon, one-way ANOVA with Tukey, Kruskal-Wallis; Pearson correlation; multiple biological replicates as specified.

• RBSDV accumulation: Viral S10 and S7-1 transcripts/proteins accumulated strongly in NIL-S but not in NIL-R at 16 dpi (e.g., S10 P = 0.042; S7-1 P = 0.015), indicating effective resistance in NIL-R.
• Transcriptomics at 16 dpi: 45 DEGs between NIL-S and NIL-R (24 downregulated, 21 upregulated in NIL-S). DEGs enriched for oxidoreductase activity and diterpenoid biosynthesis pathways. ZmGA20x7.3 (GA2ox13) was markedly upregulated in NIL-S.
• Validation of ZmGA20x7.3 induction: RT-qPCR showed significant induction in NIL-S at 16 dpi but minimal change in NIL-R; no induction at 27 dpi in either line.
• Candidate interactions: Among DEGs cross-referenced with prior Co-IP/MS, ZmGA20x7.3 showed strong interaction with ZmGDIa and P7-1, but weaker with ZmGDIa-hel (BiFC, pull-down). Other tested DEGs (ZmDAO1, ZmB1B2, ZmPP1) showed limited or no relevant interactions.
• Overexpression phenotypes: Seven independent ZmGA20x7.3-OE maize lines displayed dwarfism with progressively shortened internodes from bottom to top; significantly reduced upper internode cell length compared to B73.
• GA rescue: Exogenous GA3 fully restored plant height in ZmGA20x7.3-OE, GA1 partially, GA4 to a lesser extent. In B73, all three GAs increased height. Exogenous GAs downregulated ZmGA20x7.3 expression in OE lines but upregulated it in B73. GA1 increased ZmGA20x7.3 oligomer accumulation.
• Protein interactions and binding strength: SLC, Co-IP, pull-down, and MST showed ZmGA20x7.3 binds P7-1 and ZmGDIa strongly and ZmGDIa-hel weakly; highest affinity to P7-1, then ZmGDIa, lowest to ZmGDIa-hel. ZmGDIa exon 10 regions (and flanking segments 146–333 and 379–445) are critical for binding; helitron-derived exon 10 in ZmGDIa-hel diminishes interaction.
• Transcriptional activation: P7-1 significantly activated a 3.0-kb ZmGA20x7.3 promoter reporter in NIL-S/B73 protoplasts but not in NIL-R; P7-1-GFP transient expression induced ZmGA20x7.3 expression in NIL-S but not NIL-R; stable P7-1-OE lines showed elevated ZmGA20x7.3 expression.
• Oligomerization: ZmGA20x7.3 self-associates; GA4 enhances oligomerization (tetramers and dimers). A 3A mutant (C198A/K312A/R317A) failed to oligomerize even with GA4. Gel filtration/BN-PAGE confirmed GA4-dependent oligomer peaks (T, D) for WT but not mutant.
• Enzymatic activity linkage: ZmGA20x7.3 activity increased with GA4 concentration, saturating at 0.5 mM; oligomer (T+D) content tightly correlated with GA34 product formation (R = 0.9819). Arabidopsis OE of WT ZmGA20x7.3 caused dwarfism, whereas oligomerization-defective variant did not, indicating oligomerization is required for activity.
• Modulation by P7-1/ZmGDIa: Mixing ZmGA20x7.3 with ZmGDIa and P7-1 formed higher-order multimers (‘Mu’) and enhanced oligomerization and enzymatic activity; ZmGDIa alone had little effect; P7-1 alone reduced activity; P7-1 plus ZmGDIa significantly increased activity and oligomer content. Substituting ZmGDIa-hel suppressed oligomerization and decreased enzyme activity regardless of P7-1 presence.
• Cytology: RBSDV-infected NIL-S showed thickened cell walls, ruptured membranes, and numerous virions in upper internodes; NIL-R showed organized cytoplasm and no virions.
• Hormone profiling (60 dpi): In upper internodes, NIL-S had significantly reduced bioactive GA4 and GA1 and their products (GA34, GA5) and precursors (GA9) compared to NIL-R; GA29 increased in both lines. In upper leaves, NIL-S showed near-vanishing GA1 and significant GA4 reduction; GA9 and GA51 reduced; GA29 and GA34 barely detectable; NIL-R exhibited milder reductions.
• Auxin and cytokinins: Early (16 dpi) auxin increased in NIL-S but not NIL-R; by 60 dpi, auxin elevated in both lines, higher in NIL-R in internodes. Cytokinin levels (iP, iPR, tZ, tZR, cZ, HZD) were higher in NIL-R than NIL-S at late infection, indicating hormone imbalance underlies MRDD symptoms.
• Working model: In NIL-S, RBSDV elevates ZmGA20x7.3 expression and oligomerization; ZmGA20x7.3 acts alone or with P7-1/ZmGDIa to deplete GA1/GA4, perturbing auxin/cytokinin balance and causing MRDD. In NIL-R, ZmGDIa-hel limits ZmGA20x7.3 induction and oligomerization, maintaining GA levels and conferring resistance.

The study reveals a mechanistic framework linking RBSDV P7-1, host ZmGDIa, and GA metabolism via ZmGA20x7.3. In susceptible maize, P7-1 partners with ZmGDIa to both elevate ZmGA20x7.3 expression and enhance its GA-inactivating activity through formation of higher-order complexes that promote oligomerization, thereby depleting bioactive GA1/GA4. This GA depletion disrupts auxin and cytokinin homeostasis, leading to hallmark MRDD symptoms such as dwarfism and shortened internodes. In resistant genotypes, the helitron-derived exon 10 in ZmGDIa-hel weakens interactions with P7-1 and ZmGA20x7.3, blunting transcriptional induction and oligomerization of ZmGA20x7.3, preserving active GA levels and preventing disease development. The authors discuss that P7-1 is unlikely a direct transcription factor and may recruit host TFs to induce ZmGA20x7.3; they also note potential interactions with auxin response factors, pointing to broader reprogramming of hormone signaling. The tight correlation between ZmGA20x7.3 oligomer content and enzymatic output underscores oligomerization as the key determinant of GA2ox activity, aligning with prior findings in rice GA2oxs.

This work identifies ZmGA20x7.3 as a pivotal host factor co-opted by RBSDV P7-1 and ZmGDIa to inactivate bioactive gibberellins via GA4-dependent oligomerization, thereby driving MRDD symptom development. The helitron allele ZmGDIa-hel confers recessive resistance by weakening P7-1/ZmGDIa/ ZmGA20x7.3 complex formation, limiting ZmGA20x7.3 expression and oligomerization, and maintaining GA1/GA4 levels to mitigate disease. The findings elucidate how allelic variation at ZmGDIa modulates viral manipulation of hormone metabolism and provide a mechanistic model of MRDD pathogenesis. Future directions include identifying the transcription factors recruited by P7-1 to activate ZmGA20x7.3, dissecting the relative catalytic contributions of tetramers versus dimers, and leveraging ZmGDIa-hel and GA pathway modulation for breeding MRDD-resistant maize.

• The study could not distinguish the specific catalytic contributions of ZmGA20x7.3 tetramers versus dimers; oligomerization is linked to activity, but the active oligomer species remain unresolved.
• While P7-1 induced ZmGA20x7.3 expression in a ZmGDIa-dependent manner, the host transcription factors mediating this upregulation were not identified.
• Interactions with broader hormone signaling components (e.g., auxin response factors) are suggested by proteomics but not functionally validated in this work.