ZmDRR206 involves in maintaining cell wall integrity during maize seedling growth and interaction with the environment

doi:10.21203/rs.3.rs-2592927/v1

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ZmDRR206 involves in maintaining cell wall integrity during maize seedling growth and interaction with the environment

https://doi.org/10.21203/rs.3.rs-2592927/v1

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Plants adaptively change their cell wall composition and structure during their growth, development, and interactions with environmental stresses. Dirigent proteins (DIRs) contribute to environmental adaptations by dynamically reorganizing the cell wall and/or by generating defense compounds. Here, we established that maize DIR, ZmDRR206, mediates maize seedling growth and disease resistance by coordinately regulating biosynthesis of cell wall components for cell wall integrity (CWI) maintenance. The expression of ZmDRR206 increased rapidly in maize seedling upon pathogen infection. Both overexpression and mutation of ZmDRR206 resulted in small kernel and diminished seedling growth; while ZmDRR206-overexpression increased disease resistance, enhanced drought tolerance and reduced photosynthetic activity, thus caused the maize seedlings to show a growth and defense trade-off phenotype. Consistently, ZmDRR206-overexpression reduced the contents of primary metabolites and down-regulated the photosynthesis-related genes; while increased the contents of major cell wall components and defense phytohormones and up-regulated defense- and cell wall biosynthesis-related genes in maize seedlings grown under non-stress conditions. Furthermore, ZmDRR206 physically interacted with ZmCesA10 in yeast and in planta. Our findings unravel a mechanism that ZmDRR206 coordinately regulates biosynthesis of cell wall components for CWI maintenance during maize seedling growth, providing opportunities for breeding strong disease resistance in maize.

Biological sciences/Plant sciences/Plant stress responses/Drought

Biological sciences/Plant sciences/Plant immunity/Effectors in plant pathology

Biological sciences/Plant sciences/Plant stress responses/Biotic

cell wall integrity

cell wall biosynthesis

Dirigent protein ZmDRR206

defense response

seedling growth

The plant cell wall plays critical roles in defining cell shape and size, providing structural support for the plant body, protecting cells from invading pathogens, and acting as nodes for communication between the symplast and apoplast. Thus, cell walls are essential elements underlying growth and defense to biotic and abiotic stresses, both of which influence crop yield. Cell wall composition and structure are under active modification during various biological processes and stress responses. Maintaining cell wall integrity (CWI) is crucial for plant growth, development, and interactions with the environment; likewise, the regulatory processes controlling active and adaptive modifications of cell wall composition and structure are important for growth and defense^1,2. The developmental processes (cell elongation), abiotic stress factors (drought, salt, or cold) and the cell wall broken down by a pathogen during infection could cause in vivo cell wall damage (CWD). If the cell experiences CWD, the cell wall becomes distorted, which is detected by CWI monitoring components and leads to growth arrest. In response, the production of lignin, jasmonic acid (JA), and salicylic acid (SA) increases to activate general stress responses. Thus, higher levels of phytohormone (JA/SA) and lignin deposition are classic hallmark responses of CWI impairment. Plant cells must correct detrimental alterations to their cell wall to support growth upon a challenge to the CWI. For subsequent growth recovery, plant cells must regain their ability to loosen their walls and incorporate new polymers to avoid wall failure^3,4.

The plant cell wall is a highly dynamic structure composed of several groups of polysaccharides (cellulose, semi-celluloses, pectins), structural proteins, and phenolic compounds⁵. Flavonoids, lignins, lignans, and monolignols are major phenolic compounds that are derived from the phenylpropanoid pathway and play important roles in plant biotic defense and abiotic stress tolerance. Lignin and lignan are phenolic polymers that consist of p-hydroxyphenyl, guaiacyl, and syringyl units formed by the oxidative coupling of p-coumaryl, coniferyl, and sinapyl alcohols, respectively⁶. Bimolecular phenoxy radical coupling is especially important for the biosynthesis of lignin and lignan during vascular plant development. Oxidases, like peroxidases (PODs) and laccases (LACs), are required for the formation of the monolignol radicals and have been reported to be involved in monolignol oxidation during lignin and lignan biosynthesis⁷. Coniferyl alcohol is oxidized by peroxidase/H₂O₂ or LAC/O₂ to produce the corresponding radical and then dirigent proteins (DIRs) guide the phenoxy radical coupling reaction and mediate the stereoselective formation of either (+) or (−)-pinoresinol^8,9. DIRs are extracellular glycoproteins with a high β-strand content in all reported land plants. DIRs have no catalytic activity on their own or with a suite of potential cofactors, but direct the outcome of bimolecular phenoxy radical coupling reactions toward regio- and stereospecific products in presence of the oxidases. DIRs play a central role in plant secondary metabolism, especially the biosynthesis of lignan, flavonolignan, and alkaloids. Several genes encoding (+)- or (–)-pinoresinol-forming DIRs have been described in various plant species^10–12.

It is critical to control the spatial deposition of lignin during defense responses to biotic or abiotic stress. The participation of DIRs in this process was first supported by their co-localization with lignin initiation sites, as determined by immunohistochemistry with anti-DIR polyclonal antibodies¹³. The casparian strip (CS) is mainly composed of lignin and constitutes a physical and chemical barrier that tightly controls water and nutrient transport and provides protection against soil-borne pathogens¹⁴. The localization of CS domain proteins (CASPs) specifies the site of CS formation in cell membrane domains by recruiting proteins required for CS formation and lignification. The DIR protein AtDIR10/ESB1 is targeted to the lignification initiation sites in a CASP-dependent manner during CS formation and is required in both the early deposition of lignin patches and their fusion in generating the mature CS in Arabidopsis^15,16. The soybean (Glycine max) DIR-like protein PDH1 is involved in regulation of soybean pod dehiscence by controlling the spatial deposition of lignin¹⁷. Furthermore, Arabidopsis DIR7 plays a role in CWI maintenance, as lose function of DIR7 causes alterations in cell wall structure, impaired isoxaben (ISX, a cellulose biosynthesis inhibitor)-induced SA accumulation, and increased susceptibility to a necrotrophic fungus¹⁸. Thus, DIRs may exert essential functions in regulating cell wall modification/reinforcement during CWI maintenance.

Lignin biosynthesis plays an essential role in plant defense responses to biotic and abiotic stresses. Lignin confers stability and hydrophobicity to the plant vascular system; lignin also forms a barrier against microbial pathogens to limit the spread of pathogen-derived toxins and enzymes into the host by altering the compressibility and porosity of the cell wall^19,20. The participation of DIRs in response to pathogens that cause physical damage to the cell wall during infection has been reported in various species, whereby DIRs regulate the dynamic reorganization of the cell wall and/or generate defense compounds. The dirigent-like protein GhD2 modulates disease resistance by interacting with GhJAZ2, a member of the Jasmonate-ZIM-domain family, in cotton²¹. Various lignan stereoisomers display antibacterial activities. (+)-Gossypol is a phenolic aldehyde that plays an important role in pathogen responses, and DIR proteins participate in the formation of the phenolic terpenoid (+)-gossypol in cotton (Gossypium sp.)²². AtDIR12/DP1 is specifically expressed in outer integument cells of developing seeds and is co-expressed with the laccase gene LAC5 to regulate neolignan biosynthesis in Arabidopsis²³. In plants exposed to abiotic stress, lignification levels are frequently modulated by DIRs and peroxidases. The rise of PEROXIDASE5 and DIR2-like protein abundance in soybean coincided with elevated accumulation of H₂O₂ in roots induced by manganese toxicity²⁴. These DIR-related mechanisms may support the ability of plants to cope with various abiotic stresses, a specific response to which may be provided by the mobilization of specific POD and DIR¹³.

Maize is a major crop used for food, feed, and fuel. The more frequent occurrence of extreme weather conditions and various maize diseases often cause severe yield loss. Therefore, new maize varieties with strong disease resistance, high yield, and good quality are a constant goal for maize breeding. Several mechanisms monitor and maintain the functional integrity of plant cell walls, but knowledge of the underlying genes, molecular processes and their function in growth and defense is limited. Here, we demonstrate that the dirigent protein, DISEASE RESISTANCE RESPONSE206 (ZmDRR206), plays a role in CWI maintenance during maize seedling growth. ZmDRR206 overexpression significantly increased disease resistance against Fusarium graminearum infection and drought tolerance of maize seedlings, while simultaneously compromising kernel development and seedling growth in maize. Consistently, ZmDRR206 overexpression downregulated the expression of photosynthesis-related genes but induced that of defense-related genes, especially genes encoding enzymes for cell wall biosynthesis. We propose that ZmDRR206 involves in maintaining CWI by regulating cell wall production and remodeling during maize seedling growth and defense responses.

The expression of ZmDRR206 responds rapidly to Fusarium graminearum infection and light illumination in maize seedlings. Based on our previous studies, we discovered that expression of ZmDRR206 is significantly induced in infected seedling roots of the near isogenic lines (NILs) carrying the resistant qRfg1 (Resistance to F. graminearum) allele (R-NIL), but not in infected roots of the NIL carrying the susceptible qRfg1 allele (S-NIL). Similarly, another maize resistant allele developed from a second resistance loci (qRfg2) also possessed elevated ZmDRR206 expression in response to F. graminearum infection^25,26 (Supplementary Fig. 1a, b). Moreover, the expression of ZmDRR206 rose rapidly 6 and 18 h after inoculation with F. graminearum and was also induced rapidly by light illumination in seedlings from the inbred line LH244 (Fig. 1a, b). ZmDRR206 was annotated as an inducible pathogenesis-related (PR) gene involved in defense response to biotic stimulus and encoded a DIR family protein with a predicted molecular function of isomerase activity. Based on published high-spatial-resolution transcriptome deep sequencing datasets, we observed that ZmDRR206 is expressed in several young tissues, such as the developing endosperm and young seedling shoots, roots, young leaves, and tassels²⁷, as well as in the basal endosperm transfer layer (BETL), the embryo surrounding region, the aleurone, and the conducting zone in developing maize kernels²⁸ (Supplementary Fig. 1c-e).

To investigate the subcellular localization of ZmDRR206, we transiently infiltrated N. benthamiana leaves with Agrobacterium containing a construct expressing a ZmDRR206-GFP fusion (ZmDRR206 cloned in-frame and upstream of the green fluorescent protein [GFP] sequence) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. We detected that ZmDRR206-GFP fluorescence was associated with the cell periphery, in contrast to the even distribution of the free GFP in the cytoplasm and the nucleus. We also noticed a punctate pattern for ZmDRR206-GFP fluorescence at the cell periphery (Fig. 1c). We further prepared a specific polyclonal antibody against ZmDRR206: Tissue fractionation and immunoblotting analysis of 6-day-old wild-type (WT, LH244 inbred line) seedlings revealed a strong ZmDRR206 signal only in the membrane pellet, but not among soluble proteins (Fig. 1d). These results indicate that ZmDRR206 primarily localizes to the plasma membrane (PM).

ZmDRR206 plays a role in maize kernel development and seedling growth. To investigate the biological function of ZmDRR206, we generated six independent maize transgenic events in the LH244 background harboring a construct overexpressing the full-length ZmDRR206 coding sequence under the control of the maize Ubiquitin promoter. The transgenic events were self-crossed, and we harvested their T₄ homozygous progeny, designated here DRR-OE, for phenotyping. Compared to the fully developed smooth kernels with bright yellow color in the WT ears, the mature kernels of DRR-OE ears were opaque (soft texture) with a light-yellow color. Mature DRR-OE kernels were visibly smaller in size and shriveled at the bottom, compared to WT kernels (Fig. 2a, b). The average hundred-kernel weight (HKW), and kernel length and width of DRR-OE kernels were all significantly smaller than those of WT, with a decrease in HKW of ~ 55.6%, ~ 46.4%, and ~ 44.5% in the lines DRR-OE3, DRR-OE4, and DRR-OE6, respectively, compared to that of WT. DRR-OE kernels exhibited a shrunken and reduced endosperm, but their embryos were very similar to WT, thus exhibiting a significantly higher embryo: endosperm ratio than WT. Importantly, the germination rate of DRR-OE lines was comparable to that of WT (Fig. 2c,d; Supplementary Fig. 2a-e). These results suggest that ZmDRR206 overexpression impairs endosperm development but has no effect on embryo development or seed germination. The unloading of nutrients from the maternal placenta and their passage through the transfer tissue of BETL and the basal intermediate zone (BIZ) to the upper part of the endosperm considerably influence maize kernel weight. The BETL cells of the developing WT kernel displayed a slightly elongated shape with labyrinth-like wall in-growths, while the BETL cells of DRR-OE were shorter in length with fewer thickenings or wall in-growths. Compared to the extremely elongated BIZ cells with wall in-growths in WT kernels, the elongation was less pronounced and most of BIZ cells were short, fattened or rounded like those of the starchy endosperm cells in DRR-OE kernels (Fig. 2e). These results suggested that the DRR-OE endosperm develops dysfunctional nutrient transfer cells, especially the BETL and BIZ cells, this may retard nutrient transport from the maternal tissue to endosperm and ultimately result in impaired endosperm development.

Relative ZmDRR206 transcript levels were significantly higher in the primary roots of DRR-OE lines, as determined by reverse-transcription quantitative PCR (RT-qPCR), with a fold increase of 175.85 in DRR-OE3 and of 38.8 in DRR-OE6, compared to WT seedlings 6 days after germination (DAG). ZmDRR206 protein also accumulated to much higher levels in DRR-OE relative to WT seedlings, based on immunoblot analysis with the ZmDRR206-specific antibody (Fig. 3a, b). Both primary root length of 7-DAG seedlings and the height of 12-DAG seedlings were shorter in the DRR-OE3 and DRR-OE4 relative to WT seedlings, but not DRR-OE6 roots (Fig. 3c-f). DRR-OE plants grew more slowly than the WT during the early growth stage (~ 8 weeks) in the field. However, DRR-OE plants later resumed a WT stature, there were no significant differences between WT and DRR-OE in mature plant height. These growth dynamics (delayed early growth) suggest that ZmDRR206 may not affect the overall plant growth and development. ZmDRR206 overexpression also retarded growth of the transgenic Arabidopsis seedling, which had shorter primary root and developed relatively smaller seeds than that of Columbia wild-type seedling (Supplementary Fig. 2f,g). These results indicate that ZmDRR206 plays roles in maize kernel development and seedling growth.

A maize mutant, drr206 (EMS3-032c9c), of inbred line B73 generated by EMS mutagenesis (with the serine codon TCG at 42 aa changed into an early stop codon TAG). The mutant line was self-pollinated continuously for four generations to screen for the homozygous mutants of ZmDRR206 by phenotyping and genotyping verified by specific PCR (for primers see Supplementary Table 2) and DNA sequencing. We found that the kernel phenotype of drr206 was small and shriveled at the bottom, the HKW of drr206 was significantly smaller than that of the wild-type B73. Both the shoot and root of drr206 were significantly shorter than that of B73, indicating a slower growth rate of drr206 (Supplementary Fig. 3a-e). These further suggest that ZmDRR206 plays important roles in maize seedling growth and kernel development.

Photosynthetic activity is reduced in DRR-OE maize seedling. As the expression of ZmDRR206 was light inducible and the growth of DRR-OE seedlings was retarded, we measured chlorophyll contents and net photosynthetic rates (Pn) of DRR-OE and WT seedlings. We estimated chlorophyll contents by measuring the SPAD value at the center of the widest part of the newest expanded leaf for each seedling. As the DRR-OE3 seedlings were much smaller than WT seedlings, we focused on DRR-OE4 and DRR-OE6 seedlings. The average SPAD value of 12-DAG WT leaves was 34.3, while that of 12-DAG DRR-OE leaves was lower by ~ 22.9% (DRR-OE4) and ~ 18.6% (DRR-OE6). The average SPAD values of the 15-DAG DRR-OE leaves were also lower than that of WT. We measured Pn in the same leaf region of 15-DAG seedlings as for SPAD. The Pn values ranged from 11.3 to 12.7 µmol CO₂ m^− 2 s^− 1 in the WT and from 6.7 to 7.5 µmol CO₂ m^− 2 s^− 1 in DRR-OE seedlings, corresponding to a decrease of ~ 44.9% (DRR-OE4) and ~ 38.2% (DRR-OE6), compared to WT seedlings (Fig. 3g, h). These results indicate that the chlorophyll biosynthesis and photosynthetic activity are reduced in DRR-OE seedlings, which may contribute to the delayed growth seen with DRR-OE seedlings.

DRR-OE seedlings exhibit greater disease resistance and drought tolerance. As ZmDRR206 expression was significantly induced in the R-NIL plants after inoculation with F. graminearum, we examined the disease phenotypes of DRR-OE plants. We observed that DRR-OE seedlings exhibit higher disease resistance against F. graminearum infection, as evidenced by their lower disease severity index (DSI) relative to WT seedlings. Importantly, DRR-OE seedlings also displayed better growth of both shoots and roots after inoculated with F. graminearum, compared to WT seedlings. We confirmed this enhanced disease resistance against F. graminearum infection in field trials of mature maize plants (Fig. 4). Moreover, the DSI of the drr206 seedlings was significantly higher than that of B73 seedlings at 48 h after inoculation with F. graminearum, the disease symptoms of the mutant seedling roots were more severe with darker color or a little swollen symptom around the diseased region, compared to that of B73 seedlings, indicating the reduced disease resistance of the mutant (Supplementary Fig. 3f, g). These data demonstrate that ZmDRR206 positively regulates maize disease resistance against F. graminearum induced stalk rot.

Unexpectedly, we discovered that DRR-OE seedlings are much more tolerant to drought stress; water withholding after the two-leaf stage resulted in severe wilting of all WT seedlings at ~ 25-DAG, in contrast to the green leaves and upright stems of DRR-OE plants. We quantified this apparent tolerance to drought by scoring survival rate (SR) of DRR-OE and WT seedlings. The SR of both DRR-OE4 and DRR-OE6 seedlings was over 95% upon drought stress treatment, whereas WT seedlings only reached about 50% survival (Fig. 5a, b). We also estimated the transpiration rate (TR) at the center of the widest part of the newest expanded leaf of 15-DAG seedlings. Consistent with above observation, the TR values ranged from 0.88 to 0.96 µmol H₂O m^− 2 s^− 1 for WT seedlings, but were much lower in DRR-OE seedlings, with a range from 0.50 to 0.55 µmol H₂O m^− 2 s^− 1 in DRR-OE seedlings or a ~ 46.15% (DRR-OE4) and ~ 40.4% (DRR-OE6) decrease (Fig. 5c). Similarly, ZmDRR206-overexpression in Arabidopsis also increased the drought tolerance of the transgenic seedlings, which showed significantly increased SR and better growth behavior under severe water deficiency (Fig. 5d, e). These results indicate that ZmDRR206 plays a positive role in seedling drought tolerance.

The suppressed photosynthetic activity and the down-regulated photosynthesis-related genes in DRR-OE maize seedling. We conducted transcriptome deep sequencing (RNA-seq) to assess the effects of ZmDRR206 overexpression on gene expression in maize control seedlings (WT and DRR) and seedlings inoculated with F. graminearum (WTi). We identified 2,101 differentially expressed genes (DEGs, P-value < 0.05, absolute fold change > 2.0) by comparing gene expression between DRR-OE and WT seedlings, of which, 775 were upregulated and 1,326 were downregulated, thus implying that ZmDRR206 plays an important role in maize seedling growth under normal conditions. Statistical Gene Ontology (GO) term enrichment analysis revealed the cellular components of the proteins encoded by many downregulated DEGs were: ribosome (190 genes), ribonucleoprotein complex (208 genes), photosystem (34 genes), plastoglobules (20 genes), photosynthetic membrane (38 genes), and ribosomal subunits (153 genes). Interestingly, almost all the photosynthesis antenna protein and photosynthesis light harvesting-related genes were simultaneously down-regulated by ZmDRR206-overexpression and by pathogen infection (Supplementary Fig. 4a). Moreover, the downregulated DEGs also showed an enrichment in translation- and photosynthesis-related functional categories, such as ribosome, ribosome biogenesis, ribonucleoprotein complex biogenesis, photosynthesis antenna proteins, photosynthesis light harvesting, and photosynthesis (Fig. 6a, b), suggesting a reduced ability of DRR-OE seedling in these processes. Among the dramatically down-regulated genes in DRR-OE seedlings, the trihelix transcription factor (TF) gene ZmGT-3b exhibited an expression profile similar to that of photosynthesis-related genes. The transcripts for ZmGT-3b, PHOTOSYSTEM II3 (ZmPSII3), LIGHT HARVESTING COMPLEX II (ZmLHCII) and mesophyII7 (ZmLHCII7), were reduced to less than a fold-change of 0.4 in DRR-OE4 and DRR-OE6 seedlings, compared to WT, as seen in our RNA-seq data (Supplementary Fig. 4b) and by RT-qPCR (Fig. 6c). These results were consistent with the lower photosynthetic rates of DRR-OE relative to WT seedlings (Fig. 3h), suggesting that the retarded growth of DRR-OE seedlings may associate with the repression of growth-promoting (translation- and photosynthesis-related) genes.

We independently validated the repression of photosynthesis by Tandem Mass Tag (TMT)–labeled quantitative proteomics of whole protein extracts from maize leaves (2nd leaf of the 12-DAG seedling). We identified 376 proteins with different abundance (DAPs) by comparing protein levels in DRR-OE and WT leaf extracts, including 2.061-fold-increase of ZmDRR206 in DRR-OE relative to WT seedling. 168 DAPs were predicted to localize to the chloroplast and 108 of them showing lower abundance in DRR-OE samples. All DAPs enriched in the Kyoto Encyclopedia for Genes and Genomes (KEGG) pathways zma00195 (photosynthesis), zma00910 (nitrogen metabolism), and zma00196 (photosynthesis-antenna proteins) exhibited a lower abundance in DRR-OE relative to WT seedlings. More abundant DAPs were mainly enriched in the KEGG pathways zma00940 (phenylpropanoid biosynthesis), zma04626 (plant–pathogen interaction), zma00480 (glutathione metabolism), and zma03410 (base excision repair) (Supplementary Fig. 4c-f). Furthermore, metabolite profiling based on high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used to identify differentially abundant metabolites in the 2nd leaves of DRR-OE relative to WT seedlings at 12-DAG. The contents for all detected carbohydrates, organic acids, and four amino acids were decreased (< 0.65-fold, p-value < 0.05), while the contents of asparagine, valine, lysine, protectants (allantoin, choline, and melanin), and some secondary metabolites were increased (> 1.5-fold, p-value < 0.05) in DRR-OE relative to WT seedlings. However, the contents of different flavonoids varied, the contents of some flavones rose, and the contents of flavonols decreased in DRR-OE relative to WT seedlings. Notably, the contents of cinnamic acid (CA) showed the biggest fold-change increase (77.6-fold) (Supplementary Fig. 5). These results indicate that ZmDRR206 affects both primary and secondary metabolism in maize seedlings.

Defense-related transcriptional reprogramming is induced by ZmDRR206 overexpression. Functional annotations of the DEGs upregulated by ZmDRR206 overexpression revealed their association with oxidoreductase activity, monooxygenase activity, peroxidase activity, and ion binding (Fig. 6b), suggesting their association with important redox, synthesis and metabolism processes. KEGG pathway enrichment analysis emphasized gene functions in the biosynthesis of secondary metabolites, phenylalanine metabolism, phenylpropanoid biosynthesis, plant–pathogen interaction, and plant hormone signal transduction. Among the pathways related to biosynthesis of secondary metabolites, biosynthesis of flavone, flavonol, stilbenoid, diarylheptanoid, gingerol, benzoxazinoid, diterpenoid, flavonoid, and isoflavonoid was significantly enriched (Fig. 6a). This result was consistent with the above metabolome analysis, which showed various changes in benzoxazinoids and flavonoids (Supplementary Fig. 5). Almost all of these functional categories can be summarized in support of basal defense responses to various stresses, suggesting that ZmDRR206 is positively associated with plant defense responses.

Plants undergo a substantial transcriptional reprogramming to prioritize defense- over growth-related cellular functions upon pathogen infection. Inoculation with F. graminearum induced 1,026 DEGs in WT seedlings, including the 304 DEGs that were also induced by ZmDRR206 overexpression, pointing to commonalities in the transcriptional reprogramming induced by ZmDRR206 overexpression (DRR/WT) and inoculation (WTi/WT). DEGs induced by inoculation (WTi/WT) were also significantly enriched for GO and KEGG terms related to biosynthesis of secondary metabolites, especially biosynthesis of phenylpropanoid, stilbenoid, diarylheptanoid, gingerol, benzoxazinoid, flavonoid, diterpenoid, flavone, flavonol, carotenoid. Notably, the transcriptional reprogramming induced by ZmDRR206 overexpression (DRR/WT) was stronger for these defense response–related functional categories than that induced by inoculation of WT seedlings (WTi/WT), as indicated by their greater number of DEGs in DRR/WT relative to the WTi/WT comparison (Supplementary Fig. 6). Therefore, ZmDRR206 overexpression induces defense-related transcriptional reprogramming in DRR-OE seedlings.

ZmDRR206 may play a role in CWI maintenance by regulating cell wall biosynthesis. The defense-induced biosynthesis of lignin plays a major role in basal immunity. We thus investigated the cell wall composition of roots from 7-DAG seedlings and from 12-DAG whole seedlings. We detected a significant increase in content of acid-soluble lignin (ASL, ~ 13.0%), acid-insoluble lignin (AIL, ~ 11.1%), and lignin (~ 11.5%) in roots of 7-DAG DRR-OE seedlings, while the contents for cellulose and semi-cellulose were similar, compared with WT seedlings (Fig. 7a). By 12-DAG, DRR-OE seedlings showed greater contents for cellulose (~ 8.0%) and semi-cellulose (~ 9.7%), together with increased contents for AIL (~ 6.7%) and lignin (~ 6.1%), although ASL contents were not different, compared to WT seedlings. Arabinose levels are positively correlated with lignin levels, cellobiose can activate plant immune responses, and shifts in the xylose content of the cell wall have a deep impact on CWI^{19, 29}. Notably, we measured higher levels of cellobiose (~ 14.1%), glucose (~ 4.5%), arabinose (~ 6.5%), and xylose (~ 4.6%) in DRR-OE relative to WT seedlings at 12-DAG (Supplementary Fig. 7a, b). Furthermore, we noticed that the leaf blade and midrib were significantly thinner in 12-DAG DRR-OE relative to WT seedlings (Supplementary Fig. 7c). The observed increases in contents for the main cell wall components in DRR-OE seedlings may enhance the structural integrity and strength of the cell wall, suggesting that ZmDRR206 coordinately regulates the biosynthesis of cell wall components.

GO analysis revealed that many DEGs were significantly enriched in cell wall organization/ biogenesis–related functional categories, including xyloglucan metabolism, semi-cellulose metabolism, cell wall polysaccharide metabolism, cell wall modification, and glucan metabolism (Supplementary Fig. 8a), indicating these genes contribute to biosynthesis/modification of cell wall-related components. The influence of ZmDRR206 overexpression on cell wall composition prompted us to assess expression profiles of the related genes in DRR-OE seedlings. Consistent with the higher contents for the major cell wall components described above, transcript levels for 4 cellulose synthase (Ces) genes were significantly increased in DRR-OE relative to WT seedlings, the expression of almost all these Ces were increased specially in response to ZmDRR206-overexcpression (DRR/WT), but not to inoculation (WTi/WT), except CesLG3 (Supplementary Fig. 9a). Similarly, many genes encoding the critical enzymes in the lignin biosynthesis pathway were up-regulated in DRR-OE seedlings, including genes encoding phenylalanine ammonia-lyase (PAL), cinnamate 4-monooxygenase (C4M), 4-coumarate CoA ligases (4CLs), caffeoyl-CoA O-methyltransferases (AOMTs), cinnamyl alcohol dehydrogenases (CADs), laccases (LACs), dirigent proteins (DPs) and CASP (Supplementary Fig. 9b-d; Supplementary Table 1). Of the genes involved in xyloglucan metabolism and cell wall remodeling, including nine DEGs encoding xyloglucan endotransglucosylase/hydrolases (XTHs) and 21 DEGs encoding expansins (EXPs) were down-regulated; while many POD, UDP-glucosyltransferase (UGT), chitinase and ABC transporter encoding genes were up-regulated in DRR-OE relative to WT seedlings (Supplementary Table 1).

Consistent with the increased contents of cell wall components, the transcript levels of ZmCesA10, ZmCesA11, and ZmCesA12, which encoding cellulose synthases required for cellulose biosynthesis in secondary cell walls²⁰; as well as those of genes encoding critical enzymes in the lignin biosynthesis pathway (C4M, AOMT, CAD-bm1 [also named brown midrib], and CAD6), were all significantly up-regulated in DRR-OE relative to WT seedlings, as determined by RT-qPCR (Fig. 6d). Furthermore, ISX induced ectopic production of lignin in the upper part of WT primary root tips, but not in DRR-OE seedling roots, as revealed by staining with phloroglucinol-HCl (an indicator of secondary wall thickening) (Fig. 7b). These results indicate that ZmDRR206 may play a role in CWI maintenance by regulating cell wall production and remodeling during maize seedling growth.

The CWI maintenance system involves ion channels that constantly monitor the state of the cell wall and initiate adaptive changes in both cellular and cell wall metabolisms^1,3. Multiple ion transporters were up-regulated in DRR-OE relative to WT seedlings, including genes encoding phosphate, potassium, copper, vacuolar iron, and zinc transporters (Supplementary Table 1). We thus measured the mineral element composition of 7-DAG seedlings. Compared to WT seedlings, DRR-OE seedlings accumulated more magnesium (Mg), potassium (K), sodium (Na), and phosphorus (P), but presented a lower abundance of aluminum (Al) and iron (Fe). The contents for copper (Cu) and zinc (Zn) were comparable across seedlings (Fig. 7c, d), indicating that cellular osmotic conditions (ion contents) are altered in DRR-OE seedlings. These results further suggest that ZmDRR206 may play a role in CWI maintenance.

The altered biosynthesis of the defense-related phytohormones and the constitutive expression of defense-related genes in DRR-OE seedlings. The altered cell wall composition observed in DRR-OE seedlings prompted us to check the transcript levels of JA/SA and ethylene biosynthesis genes in DRR-OE seedlings, as the impairment of CWI triggers responses that include the activation of defense gene transcription; JA, SA, and/or ethylene production; and lignin accumulation^30,31. Although it is well-known that most LOXs could be induced by JA, eight lipoxygenase (LOX) genes were found to be specially up-regulated in DRR-OE relative to WT seedlings, some of them might potentially synthesize JA; while the upregulation of the genes encoding allene oxide synthase (AOS) and allene oxide cyclase (AOC) did not reach statistical significance. Four PAL genes were up-regulated in DRR-OE seedlings, together with the significantly elevated CA content in DRR-OE (Supplementary Fig. 8b; Supplementary Table 1), indicating that both JA and SA biosynthesis are induced. Consistently, the contents of JA, JA-Ile (the bioactive form of JA), 12-oxo-phytodienoic acid (OPDA, the precursor for JA biosynthesis), and SA were all significantly increased, while ACC (the precursor for ethylene biosynthesis) content was significantly decreased in DRR-OE relative to WT seedlings (Fig. 8a). Moreover, JA/SA-regulated and/or defense-related genes were constitutively up-regulated in DRR-OE seedlings, including jasmonate-regulated genes (JRGs), jasmonate-induced protein genes (JIPs), vegetative storage protein genes (VSPs), pathogenesis-related genes (PRs), disease resistance genes and chitinase genes (Supplementary Table 1). We confirmed the up-regulated expression of several defense-related genes in DRR-OE relative to WT seedlings by RT-qPCR, including two WRKY TF genes (WRKY11 and WRKY69), four PR genes (PR1, PR10a, PR10b, and PR3), three chitinase genes (CHN5 [chitinase chem5], AEC [Acidic endochitinase], and BEC [Basic endochitinase A]), and five JA-regulated genes (two JIPs, VSPpni288, VSP2, and LOX2) (Fig. 8b). Furthermore, ZmDRR206 overexpression significantly upregulated genes belonging to multiple TF families such as MYB (eight genes), WRKY (nine genes), basic helix-loop-helix (bHLH, eight genes), ERF (13 genes), basic leucine zipper (bZIP, five genes), and NAC (seven genes). Other genes encoding growth-promoting TFs were downregulated in DRR-OE relative to WT seedlings, including GATA genes, TCP genes, PRE3 genes (Supplementary Table 1). These altered patterns of gene expression further suggest that ZmDRR206 plays a role in CWI maintenance and contributes to the altered growth and stress response of DRR-OE seedlings.

ZmDRR206 interacts with a cellulose synthase subunit ZmCesA10. To further investigate the molecular mechanism of ZmDRR206, we searched for its interaction partners. The STRING database (https://string-db.org/cgi) suggested several candidates that we compared to our quantitative proteomics dataset (Supplementary Fig. 4g). We also checked the expression profiles of these candidate genes in qTeller (https://qteller.maizegdb.org/rna_data_sources.php) and in our RNA-seq data, culminating in the selection of three candidates: ZmCesA10, ZmDIN1 (DARK-INDUCED1, a thiosulfate sulfur transferase), and ZmRin1 (Ribonuclease 1). We assessed their potential interaction with ZmDRR206 in a split-ubiquitin membrane-based yeast two-hybrid system (DUALmembrane system) and by luciferase complementation imaging (LCI) assay. ZmDRR206 was fused to the C-terminal half of ubiquitin (Cub), and partial ZmCesA10 (CesA10p, a peptide containing the first four transmembrane domain shared by all the four proteins encoded by the splicing variants of ZmCesA10), ZmDIN1, or ZmRIN1 was fused to the mutated N-terminal half of ubiquitin (NubG). As the negative controls, the co-transformation of DRR206-Cub with the NubG empty vector didn’t enable the yeast cells to grow on the selective medium. In contrast, the yeast cells co-transformed with DRR206-Cub and NubG-CesA10p; DRR206-Cub and NubG-DIN1; or DRR206-Cub and NubG-RIN1 constructs, were able to grow on the selective medium, indicating that DRR206 and CesA10p, DRR206 and DIN1, DRR206 and RIN1 interacted in yeast to activate the expression of the reporter gene for growth selection (Fig. 9a). In the LCI assay, we cloned the coding sequences of the potential interactors in-frame and upstream of the sequence of the N-terminal half of firefly luciferase (nLUC, in the JW771 vector) to produce CesA10p-nLUC, DIN1-nLUC, and RIN1-nLUC. We also cloned the coding sequence of ZmDRR206 into JW772 (harboring the sequence for the C-terminal half of luciferase, cLUC) to produce cLUC-DRR206. We detected strong luminescence signals when the pairs of constructs cLUC-DRR206 and CesA10p-nLUC; cLUC-DRR206 and DIN1-nLUC; or cLUC-DRR206 and RIN1-nLUC were co-infiltrated into N. benthamiana leaves, but not their negative control construct combination (Fig. 9b), indicating the interaction occurred between DRR206 and CesA10p, DRR206 and DIN1, DRR206 and RIN1 in planta. The interaction of ZmDRR206 and ZmCES10p was further confirmed by Co-IP assay in vivo (Fig. 9c). Moreover, in contrast to the random distribution of the free GFP or mCherry signal in the cytoplasm and nucleus, both DRR206-GFP and CesA10p-mCherry signal associated with the cell periphery, CesA10p-mCherry alone accumulated into large spots in the cell periphery (suggesting its association with specific subdomain of the PM), while DRR206-GFP signal could co-localize with CesA10p-mCherry signal in the cell periphery of the epidermal cells and DRR206-GFP could disrupt the accumulated large spot induced by CesA10p-mCherry alone (Fig. 9d).

Furthermore, we observed a marked co-expression between ZmDRR206 and ZmCesA10 during maize growth and development, for example, ZmDRR206 and ZmCesA10 were abundantly expressed in young shoots, roots, and leaves (leaf1 and leaf2)²⁷; the expression of both genes was induced by F. graminearum inoculation after 6 h in 5-DAG roots of the resistant NIL, but not in the susceptible NIL²⁵. Moreover, ZmDRR206 and ZmCesA10 were specially expressed to comparable levels in crown root nodes, non-pollinated internodes at 24 DAP, V9 eleventh leaves, V9 immature leaves, V9 thirteenth leaves, 5-day-old primary roots, 5-day-old root cortex, and 7- to 8-day-old secondary roots, based on a survey of RNA-seq data through qTeller (Supplementary Fig. 10). These results further suggest a possible interaction between ZmDRR206 and ZmCesA10 occurs in vivo.

The plant cell wall is a complex, dynamic molecular network whose composition and structure are under constant change during development and in response to stress. Plant cells monitor the status of their cell walls with various types of sensors and receptors at the plasma membrane, some of which may interact with cell wall components to coordinate mechanical changes in cell wall structure and cellular responses⁴. The disruption of CWI results in a variety of compensatory reactions, including ectopic deposition of lignin³⁰, ROS production³¹, alteration of other cell wall components³², and an increase in JA and ethylene production³³. The reallocation of energy to defense generally comes at the expense of plant growth due to competition for limited resources between growth and defense responses³⁴. The observed stunted growth of many cell wall mutants associated with attenuated cell wall biosynthesis may partially reflect responses mediated by constitutive activation of defense pathways, and the ensuing trade-off between growth and defense, rather than being directly caused by a physically weakened cell wall^19,35,36. Cell wall strengthening can be activated by loss of CWI via the mechanisms that monitor cell wall status. DIRs have been suggested to represent one set of potent effectors acting downstream of the CWI signaling cascade by mediating the spatial control of lignin deposition during growth and stress responses^13,31.

In this study, we demonstrated that ZmDRR206 actuates CWI maintenance and confers disease resistance in maize. The expression of ZmDRR206 was induced in multiple maize lines with different background upon pathogen infection, suggesting that ZmDRR206 exerts a similar conservative function in enhancing disease resistance as its soybean ortholog¹². There are three major findings in this study. First, we identified ZmDRR206 as a regulator of maize seedling growth and basal immunity: The abundance of this protein was positively correlated with maize disease resistance by regulating cell wall biosynthesis and defense responses, while negatively associated with photosynthesis, thus shedding light on how maize integrates growth, overall cell wall biosynthesis, and adaptation to biotic stress. Second, we showed that ZmDRR206 plays a role in CWI maintenance by coordinately regulating the biosynthesis of cell wall components. It is well known that defense-induced lignin plays a major role in basal immunity to promote plant fitness. However, gene(s) that coordinately regulates biosynthesis of cell wall components remains obscure. ZmDRR206 overexpression increased the contents of major cell wall components and conferred resistance to ISX treatment. An interaction between ZmDRR206 and ZmCesA10 was also established. Third, we determined the negative effect of ZmDRR206 overexpression on physiological processes like photosynthesis and translation during maize seedling growth, this might associate with the interaction between ZmDRR206 and different protein partners.

ZmDRR206 involves in CWI maintenance during maize seedling growth and defense responses. Plant cell wall modifications through mutation or overexpression of cell wall-related genes substantially influence disease resistance and/or tolerance to abiotic stresses^37,38. The observed disease resistance phenotypes in the mutants/transgenic plants with wall alterations are thought to be caused by the activation of defense signaling pathways, rather than as an inability of pathogens to overcome these modified wall composition/structures^20,39. Cellulose affects many aspects of plant life and fitness through its central role in determining the mechanical properties of plant cell walls. A functional cellulose synthase complex (CSC) requires the cooperation of at least three distinct CesA proteins for the biosynthesis of both primary and secondary cell walls^40–42. Arabidopsis CesA8, CesA7, and CesA4 are the secondary cell wall–specific cellulose synthase catalytic subunits; their respective mutants, irregular xylem1 (irx1), irx3, and irx5, display collapsed xylem, altered CWI and enhanced disease resistance against multiple pathogens that are associated with altered expression of genes related to CWI^{43, 44}. The constitutive activation of immune responses in irx1/3/5 plants probably explains their trade-off phenotypes (dwarf plant, reduced seed yield and higher disease resistance). Likewise, all mutants identified in OsCesA4, OsCesA7, and OsCesA9, which encode the functional orthologs of the Arabidopsis enzymes⁴⁵, share a brittle culm (bc) phenotype, lower cellulose contents in their secondary wall and accumulate JA and ethylene, and abnormal plant growth, characterized by a dwarf stature with small leaves and withered leaf tips^46–48. DRR-OE seedlings also displayed diminished growth; enhanced disease resistance; the concomitant rise of cellulose, semi-cellulose, and lignin contents and increased JA and SA levels, compared to WT seedlings (Figs. 3–8). Consistently, CesA genes and a subset of secondary metabolite biosynthesis-related genes, especially genes encoding enzymes for lignin biosynthesis, including ZmCesA10, ZmCesA11, and ZmCesA12, were constitutively upregulated in DRR-OE relative to WT seedlings (Fig. 6d). ZmCesA10 is an ortholog of OsCesA7. We established that ZmDRR206 physically interacts with ZmCesA10 and that their encoding genes are co-expressed in various maize tissues (Fig. 9; Supplementary Fig. 10a-c). ISX is a frequently used tool to impose cell wall stress in plant CWI maintenance research, as ISX impairs the function of CesA enzymes to induce the reorganization of primary and secondary cell walls and altered wall compositions with lower cellulose biosynthesis, which causes CWD and subsequent CWI alterations that activate immune responses, like the ectopic biosynthesis and deposition of lignin, and increased production of JA and ethylene. DRR-OE seedlings had higher contents of cell wall components and were resistant to ISX treatment (Fig. 7a, b), the resistance to ISX induced cell wall stress of DRR-OE seedlings may associate with their reinforced or strengthened cell wall by higher contents of cell wall components.

The CWI maintenance has been identified to be closely interconnected with mechanosensing and turgor monitoring for key processes underlying plant development and stress response. Ion channels and ion transporters are involved in monitoring turgor pressure levels. Increasing cytosolic ion concentrations by taking up inorganic ions from the environment via transporters and channels is a cost-effective method for intracellular osmotic adjustment in plants⁴⁹. Compared to WT seedlings, DRR-OE seedlings accumulated more Mg, K, Na, and P (Fig. 7c, d). Potassium (K⁺) is the most abundant cation and is critical for the adaptive responses of plants to various abiotic or biotic stresses, as increased K⁺ uptake confers higher levels of drought tolerance⁵⁰. Fe is involved in various chelation and oxidation/reduction steps that affect ROS production because it is a component of all photosystems and a critical redox-active metal ion in photosynthetic electron flow. As the first barrier for metal ions, lignin biosynthesis is associated with heavy metal absorption, transport, and tolerance in plants. Lignin binds to multiple heavy metal ions and reduce their entry into the cytoplasm due to its numerous functional groups (e.g., hydroxyl, carboxyl, and methoxyl)⁵¹. The reduced Al and Fe contents in DRR-OE versus WT seedlings might be associated with increased lignin biosynthesis. Together with the altered cell wall composition and the cellular osmotic conditions, the upregulation of cell wall biosynthesis-related genes, the constitutively activated defense response, the trade-off phenotype of DRR-OE seedlings, the interaction between ZmDRR206 and ZmCesA10, we propose that ZmDRR206 coordinately regulates the biosynthesis of cell wall components and plays a role in CWI maintenance during maize seedling growth and defense responses.

Cell wall not only strengthens the plant body, but also has key roles in plant growth, intercellular communication and defense¹³. The disruption of the coordinate biosynthesis of cell wall components would cause cell wall stress, disrupted CWI and the imbalance of growth and defense, that is, too much or deficient cell wall components may inappropriately reinforce or weaken cell wall and cause cell wall stress, disrupt CWI and cause similar growth defects. The similar defects in maize kernel development and seedling growth caused by ZmDRR206 overexpression (Figs. 2, 3) or mutation (Supplementary Fig. 3), suggesting that the accurate regulation of CWI is critically important for these development processes. The reduced disease resistance of the mutant drr206 might associate with the role of ZmDRR206 in basal defense response, similar to the increased susceptibility to a necrotrophic fungus of the Arabidopsis DIR7 mutant¹⁸.

ZmDRR206 overexpression activates constitutive defense responses in maize seedling. The biosynthesis of cell wall components is coordinately regulated, deposition of cellulose and lignin in cell walls are vital for growth and defense responses^52,53. Lignin contributes to pathogen resistance, while pathogens can target the host proteasome to modify the cell wall secondary metabolism to facilitate their invasion. CAD is the final enzyme in the monolignol biosynthetic pathway and thus directly affects lignin accumulation. Rhizoctonia solani can target ZmCAD for ubiquitination and degradation by interacting with the F-box protein ZmFBL, resulting in reduced lignin accumulation and susceptibility to banded leaf and sheath blight⁵⁴. Chitinases are classic PR proteins involved in non-host-specific defense and are thought to be involved in cell wall modification and activation of ethylene production⁵⁵. ZmDRR206 over-expressors had enhanced cell wall biosynthesis; increased JA, JA-Ile and SA levels; and constitutive expression of many defense-related genes, including multiple PAL, PR, chitinase and CAD encoding genes and JA regulated genes (Fig. 8b, Supplementary Table 1). However, it was unexpected to find the lower ACC level in DRR-OE seedlings, it is possible that ACO activity might be higher than ACC activity, thus leading to more ethylene production and stronger signaling (the up-regulated 13 ERFs) in DRR-OE relative to WT seedlings (Fig. 8a, Supplementary Table 1). Melatonin plays an important role in ameliorating various common abiotic and biotic stresses, including cold, drought, heavy metals, salt and pathogen attack⁵⁶. The contents of the three protectants, allantoin, choline, and melanin, were all increased > 1.5-fold in DRR-OE relative to WT seedlings (Supplementary Fig. 5). Collectively, ZmDRR206 overexpression activated a constitutive defense response, visible as the slow growth phenotype of DRR-OE seedlings from the growth–defense trade-off.

ZmDRR206 may affect different physiological pathways by physically interacting with various proteins. Indeed, we determined that ZmDRR206 interacts with ZmDIN1, which is a senescence-associated dark-inducible protein. DIN genes constitute the basal immune responses used by cells to cope with stresses imposed by senescence, pathogen infections and by SA, MeJA, darkness, or photosynthesis inhibitors⁵⁷. A common theme to these stress conditions is cellular damage caused by oxidative stress, suggesting there may be overlapping factors that may participate in various stress-responsive pathways. Arabidopsis DIN6 and DIN11 are key regulators during systemic virus infection⁵⁸. Tobacco (Nicotiana tabacum) Ntdin is involved in sulfur and nitrogen metabolism due to its relationship with nitrate reductase activity and molybdenum cofactor (Moco) biosynthesis. Ntdin expression is also upregulated by both ABA and IAA, likely because the biosynthesis of these phytohormones requires Moco⁵⁹. The physical interaction between ZmDRR206 and ZmDIN1 may thus modulate various aspects of the constitutive defense response in DRR-OE seedlings.

The possible role of ZmDRR206 in photosynthesis and translation. Photosynthesis affects the cellular ability to initiate a defense response by generating ROS and immune response-related signals like SA. Pathogen infection represses photosynthesis, affects primary metabolism, slows down plant growth, and modifies secondary metabolism toward defense responses. The products of photosynthesis (sugars) can activate defense-related responses and modulate key signaling elements of defense phytohormone pathways in an attempt to strike a precise balance between growth and defense responses⁶⁰. An effect on photosynthetic properties by cell wall dynamics has been reported. Indeed, diffusion of CO₂ from the leaf intercellular air space to the site of carboxylation is a potential trait for increasing photosynthetic efficiency and crop productivity. Cell wall characteristics like thickness and porosity have been reported to be key traits determining mesophyll conductance (g_m) and photosynthesis, as dynamic changes in cell wall composition and rearrangements of cell wall components have been shown to affect g_m under nonstress conditions^61–64. Modulation of photosynthesis by cell wall modifications has been demonstrated in rice mutants. For instance, knockout mutants in Cellulose synthase-like family 6 (OsCSLF6) are disrupted in cell wall mixed-linkage glucan (MLG) production and have lower g_m compared to the WT, suggesting that a change in cell wall properties influences the diffusivity and availability of CO₂⁶⁵. The rice fragile culm 24 (Osfc24) mutant is dwarf and brittle, whose cell wall composition is altered, with lower contents of cellulose and pectin, but higher levels of semi-cellulose and lignin. The Osfc24 mutant has pale leaves with lower chlorophyll contents, as well as diminished photosynthetic activity⁶⁶. Changes in pectin physicochemical properties induce modifications in wall architecture by rearranging cell wall compounds. The Arabidopsis mutants atpme17.2 (pectin methylesterase17.2) and atpae11.1 (pectin acetylesterase11.1) are characterized by a lower proportion of cell wall pectin, and decreased g_m and photosynthesis⁶⁷. Collectively, these observations suggest that cell wall modifications play a substantial role in determining photosynthetic output. ZmDRR206 overexpression repressed the expression of photosynthetic genes and activated the expression of cell wall formation genes, resulted in the contents of cell wall components to be higher, while chlorophyll contents and photosynthesis activity to be lower in DRR-OE relative to WT seedlings (Figs. 3, 6), suggesting that ZmDRR206 may affect chlorophyll biosynthesis and photosynthesis by regulating cell wall biosynthesis.

ZmRIN1 is annotated as a ribonuclease with endoribonuclease and RNA binding activity, is secreted into the extracellular region and involved in RNA catabolism, and plays pivotal roles in RNA maturation and decay, including ribosome RNA processing⁶⁸. The physical association between ZmDRR206 with ZmRIN1 may explain the downregulation of DEGs enriched in ribosome- and translation-related functional categories in DRR-OE relative to WT seedlings, reflecting a negative association between ZmDRR206 and translation (Fig. 6a, b). Ribonucleases have been proposed to provide cells with the means to recognize and destroy unwanted genetic material⁶⁹. Small RNA–mediated sequence-specific cleavage is utilized in both prokaryotes and eukaryotes to combat virus infection via the double-stranded RNA binding domain of ribonucleases. Moreover, ribonucleases are also an essential component for the generation of microRNAs (miRNAs), which are small noncoding RNAs that fine-tune protein abundance via translational repression in both the plant and animal kingdoms. miRNAs also act as a sequence-specific guides for the RNA-induced silencing complex by binding to their cognate mRNA targets and inducing their cleavage⁶⁸. ZmDRR206 may thus be involved in the suppression of ribosome and translation activity through its interaction with ZmRIN1.

Together, our results indicate that ZmDRR206 likely acts as a conserved component in maize seedling growth and defense responses to external biotic/abiotic stresses through its physical interactions with different protein partners that participate in various biological processes. We thus proposed a working model for ZmDRR206 in maize seedling growth and defense response: ZmDRR206 may coordinately regulate cell wall biosynthesis for CWI maintenance and defense response by physically interacting with ZmCesA10 and ZmDIN1, thus optimizing the temporal and spatial expression of growth promotion (photosynthesis and translation), cell wall biosynthesis, and defense-related genes to coordinate metabolism during seedling growth and defense response to external environmental stress (Figure. S11). More work is needed to understand the roles of the interaction between ZmDRR206 and these proteins in plant growth and defense responses.

Plant materials. To generate the transgenic, ZmDRR206 overexpression maize plants, the full-length ZmDRR206 cDNA was obtained by RT-PCR and was cloned into a pBXCUN-derived binary vector to generate the construct pUbiquitin: ZmDRR206, in which ZmDRR206 was driven by the maize Ubiquitin promoter (primers were listed in Supplementary Table 2). The plasmids were transformed into the Agrobacterium strain EHA105, and then into the immature embryos of the Zea mays L. LH244 inbred line (WT, used as the control in the afterward experiments) by Agrobacterium mediated transformation. Six independent ZmDRR206 overexpression maize transgenic events were generated, and their T₄ homozygous progenies were developed by continuous selfing and named as DRR-OE. The maize plants (WT and DRR-OE) were cultured in Shangzhuang experimental station, Beijing of China (39°54’N, 116°25'E) in summer; and were grown in Sanya, Hainan of China (18°30'N, 108°81'E) in winter, every year.

F. graminearum inoculation, disease severity scoring and seedling phenotypic analysis. The fungal pathogen F. graminearum preparation and inoculation in the field were done as previously described ⁷⁰, the maize plants were grown and inoculated in Shangzhuang experimental station, Beijing of China in 2017, 2018. The young seedling inoculation on primary roots of 5- DAG seedlings and disease severity scoring were done as previously described ²⁶. The primary roots with typical symptoms were scored at 48 hour-after-inoculation (hai). The seedlings (WT and DRR-OE) were cultured in controlled growth room under conditions of 28/22°C (day/night) at a light intensity of 500 µmolm^− 2s^− 1 (16-h-light/8-h-night) and ~ 50% relative humidity. Three replicates were set for each genotype with about 15 seedlings per replicate. For analysis of ZmDRR206 expression level, the WT seedlings were inoculated with F. graminearum at 5-DAG, then cultured in controlled growth room under light and sampled at 0 h, 6 h and 18 h after inoculation. For comparing seedling root growth rate, the length of the 7-DAG young seedling primary roots cultured with paper-rolling were measured and shoot growth rate was measured with the soil-growth young seedlings at 12- or 15-DAG. The experiments were repeated for at least three times.

Drought stress treatment. For drought stress treatment, maize seedlings (DRR-OE and WT) were grown in controlled growth room conditions of 28/22°C (day/night) at a light intensity of 500 µmolm^− 2 s^− 1 (16-h-light/8-h-night) and 40–50% relative humidity, under well-watered conditions until drought treatment. Drought stress was imposed on the two-leaf seedlings by withdrawing water supply and keeping the seedlings under observation for the following 15 days, at the time, indications of severe withering symptoms (the leaves and stems turned to be soft, dried and drooping) were visible in all of the WT seedlings, then the seedlings were re-watered. Measurements were made at 6 days after re-watering. The number of survived seedlings and the total seedling number were used to obtain the survival rate. For Arabidopsis dehydration assay, the 7-day-old seedlings grown on 1/2MS medium were transferred to soil to grow for 14 days under 8 h light/16 h dark conditions, 22°C, and subjected to drought stress for an additional 10 days. Then the plants were re-watered, and the number of plants that survived the stress and recovered their developmental phenotype was scored at 5 days after re-watering. These experiments were repeated for three times.

Subcellular localization analysis. To investigate the subcellular localization of ZmDRR206, the coding sequence of ZmDRR206 was cloned into a pCaMV35S: GFP vector to fuse in frame with the GFP-coding sequence and under the CaMV35S promoter for constructing pCaMV35S: ZmDRR206-GFP vector. Partial cDNA of ZmCesA10 that encoding a peptide containing the first four transmembrane domain (shared by all the four proteins encoded by the splicing variants of ZmCesA10, corresponding to the 752aa to 997aa of the protein with 1087aa) was obtained and fused in frame with the mCherry (or Myc)-coding sequence and under the Super promoter for constructing pSuper: ZmCesA10p-mCherry (-Myc) vector (primers were listed in Supplementary Table 2). Agrobacterium strain EHA105 containing the vector was cultured at 28°C overnight. Agrobacterium cells were harvested by centrifugation and resuspended with buffer (10mM MES, pH 5.7, 10mM MgCl2, and 200 mM acetosyringone) at OD₆₀₀ = 0.8. Leaves of 5-week-old soil-grown N. benthamiana were infiltrated with Agrobacterium cultures, with/without combination for co-infiltration, carrying the vector pCaMV35S: GFP, pCaMV35S: ZmDRR206-GFP, pSuper: ZmCesA10p-mCherry for expressing GFP, ZmDRR206-GFP, ZmCesA10p-mCherry respectively. The plants were incubated under 16h light/8h dark at 25°C, light intensity of 500 µmolm^− 2 s^− 1 and ~ 50% relative humidity in growth chambers. The GFP or mCherry fluorescence signals were detected 2-day-past-infiltration. Excitation wavelengths of GFP and mCherry were 488 nm and 561 nm, respectively. The emitted signals of GFP and mCherry were collected between 500 and 535 nm and between 580 and 620 nm, respectively. Fluorescence images were examined and taken with LSM 880 confocal laser microscope systems and images were processed using LSM microscope imaging software. The Agrobacterium tumefaciens strain containing pCaMV35S: ZmDRR206-GFP construct was transformed into Col plants using the floral dip method, to generate the ZmDRR206-overexpression transgenic Arabidopsis plants.

Measurement of leaf photosynthetic parameters. WT and DRR-OE seedlings were grown in the field under natural condition in May, 2019 or grown in a glass greenhouse with a controlled temperature of 25°C, ~ 50% relative humidity, and the light intensity could reach to 1100 µmol m^− 2s^− 1 from 9:00 am to 14:00 pm in sunny days. The seedlings at three leaves stage grown under above two conditions were used for the photosynthetic parameter measurements in a sunny day as previously described ⁷¹. The middle widest part of the latest expanded leaf of every seedling, that is the 2nd leaf of the12- DAG seedlings and the 3rd leaf of the 15-DAG seedlings, was used for measuring the soil plant analysis development (SPAD) value (leaf chlorophyll content), with a SPAD meter (SPAD-502 Plus, Konica Minolta, Inc. Tokyo, Japan) under a saturating actinic light (660 nm) with an intensity of 1100 µmol m^− 2s^− 1. The net photosynthetic rate (Pn) and transpiration rate (Tn) were evaluated on the middle part of the latest expanded leaf (the 3rd leaf of the 15-DAG seedling) that is fully light-exposed with a Li-6400XT Portable Photosynthesis System (LI-COR, LiCor Inc., Lincoln, NE, USA), recorded at a saturating actinic light (660 nm) with an intensity of 1100 µmol m^− 2s^− 1, at the time from 09:00 to 12:00 in the morning, following the manufacturer’s instructions. Replicates were taken for each genotype.

Kernel phenotype and cytological observation. For the phenotypic analysis of maize mature kernels, the kernels from the middle region of each well-filled ears with uniform kernel set were used for phenotyping and kernel trait measurement, and 100 kernels were measured for each ear. Kernel length and width were measured using an image analysis method provided with SC-E software (Hangzhou Wanshen Detection Technology). Three replicates were collected for each genotype. For cytological observations, the kernels at 16-day-after-pillination (DAP) were divided into three parts, the central part of the kernel with embryo was fixed in 50% (v/v) ethanol, 5% (v/v) acetic acid, and 3.7% (w/v) formaldehyde over 12 h at 4°C, followed by dehydration and embedding in paraffin. Longitudinal sections from the middle part of kernels were cut using a Leica Ultracut (Leica, Germany) rotary microtome and stained with Periodic acid Schiff (PAS) as previously described⁷².

RNA-seq and RT-qPCR. For RNA-seq analysis, the seedlings were cultured under the condition described, the 5-DAG seedlings with a primary root length about 6–8 cm were selected and inoculated at 10:00 am, then cultured under light for the beginning 10 h and turned to dark for 8 h; sampled in the next day at 18 hours after inoculation. The non-inoculated seedlings cultured under the same condition were collected at the same time. The seedlings without the part around the kernel were frozen in liquid nitrogen and used for RNA extraction and deep sequencing in 2016. Total RNA was extracted and the integrity and concentration of RNA samples were evaluated. RNA-seq libraries were constructed according to the protocol of the VAHTS mRNA-seq Library Prep Kit (Vazyme, Nanjing, China) and sequenced on the Illumina HiSeq 2500 platform to generate 150-bp paired-end reads. Read quality was checked and clean reads were mapped to the B73 reference genome (RefGen_v4, AGPv4). Calculation and normalization of gene expression were based on the reads per kilobase per million mapped reads calculation (FPKM) using Cufflinks. FPKM values were calculated to identify differentially expressed genes (DEGs) (P < 0.05, absolute fold change > 2.0). GO enrichment analysis of the DEGs was performed using the agriGO enrichment analysis tool (http://bioinfo.cau.edu.cn/agriGO).

For quantitative real time reverse transcription PCR (RT-qPCR) analysis, WT seedlings were grown in the dark under growth room conditions described for 5 days and transferred to light for 1 h and 2 h to analyze light-responsive expression of ZmDRR206. The seedlings were cultured under growth room conditions described for 6 days and used for expression analysis of specific genes. Total RNAs were extracted from the young seedling tissues with RNAiso Plus (Takara Bio), One microgram of total RNA was incubated with DNase I at 37°C to remove the genomic DNA, and then the prepared RNA was used for first-strand cDNA synthesis using PrimeScript RT Master Mix (Takara, Dalian, China). Then qPCR assays were performed with the TB Green Premix Ex Taq kit (Takara) following the manufacturer’s protocols on a 7500 Real-Time PCR system (Applied Biosystem) to detect specific genes. The used primers were listed in Supplementary Table 2. Relative expression of gene was calculated using the relative quantification method with maize GAPDH (accession no. X07156) and ZmTubulin1 as the endogenous control. The variation in expression was estimated using three independent biological replicates.

Analysis the contents of cell wall components. The 7-DAG seedling roots and 12-DAG maize seedlings, cultured under conditions described above, were harvested, dried and homogenized to a fine powder using a mixer mill at 25Hz for two minutes. The powdered root or seedling tissues were prepared to exclude protein and other UV-absorbing materials done according to the description by Moreira-Vilar et al⁷³ and sequentially used for measuring the contents of the major cell wall components. For quantification of lignin by the Klason method with two stage H₂SO₄ hydrolysis: Protein-free cell wall sample was digested (72% H₂SO₄, 47^oC, 7 min) under vigorous stirring, then incubated for 60 min at 30^oC and diluted the concentration of H₂SO₄ to 4%. After complete digestion, the sample was autoclaved (121^oC, 1 atm, 30 min), followed by filtration through filter paper to separate the soluble and insoluble fractions. The calculation of the AIL and ASL was done as previously described⁷³. The acid soluble lignin was analyzed on a UV-Visible spectrophotometer (TU-1901), run a background of 4% sulfuric acid. The insoluble fractions were used to analyze for acid insoluble lignin: Use deionized water to totally transfer all remaining solids and rinse the solids with fresh deionized water. The crucible and acid insoluble residues were dried and recorded the weight, then placed the crucibles and residue in the muffle furnace (Vulcan 3-550) at 575 ± 25°C for 24 hours. Removed the samples from the oven and cool in a desiccator. Recorded the weight of the crucible and dry residue for acid insoluble lignin analysis. The lignin content was determined by the sum of the insoluble and soluble lignin and was expressed as g.kg^− 1 dry weight of the tissue (g/kg.DW). For analysis of cellulose and semi-cellulose content, the de-starched alcohol insoluble residues were hydrolyzed by trifluoroacetic acid. The supernatants were separated and the remains were treated in Updegraff reagent and hydrolyzed with 72% sulfuric acid. The sugars in the supernatant were separated using an SP0810 column (Shodex) on a UHPLC system (Agilent-1260). The content of the detected sugars was calculated based on standard curves of glucose, xylose, mannose, galactose, arabinose and was used for determination of cellulose and semi-cellulose content expressed as g.kg^− 1 dry weight of the tissue. Error bars indicate SD of three replicates.

Measurement of the mineral element content. To measure the contents of the mineral elements in maize seedlings, the WT and DRR-OE seedlings at 7-DAG were sampled and dried at 60°C to constant weight for 24 h. About 0.3g dry weights of the seedlings were digested with HNO₃ at room temperature overnight. The digested solution was then diluted with deionized water and analyzed for the concentrations of the mineral elements, using atomic absorption spectrophotometry. The working parameters of ICP-AES (Model ICAP-6300, Thermo Electron, Beverly, MA, U.S.A.) were as follows: RF power: 1150 W; peristaltic pump speed: 50 r/min; auxiliary gas flow rate: 0.5 L/min; atomizer gas flow rate: 0.7 L/min. The wavelength range of the ICAP-6300 is 166-847nm. The detection wavelength for the specific mineral is as following: Al, 396.1nm; Cu, 324.7nm; Fe, 259.9nm; K, 766.4nm; Mg, 279.5nm; Na, 589.5nm; P, 213.6nm; Zn, 213.8nm. The contents were then calculated with the concentrations of the mineral elements in the diluted solution and tissue dry weights. Replicates were taken for each genotype.

Measurement of phytohormones. The endogenous SA, JA and ACC measurement assay was performed by Wuhan Greensword Creation Technology Co. Ltd., (Wuhan, China) (http://www.greenswordcreation.com/index.html) based on UHPLC-MS/MS analysis (Thermo Scientific Ultimate 3000 UHPLC coupled with TSQ Quantiva). To measure the endogenous concentrations of SA and JA, 500-mg samples of 6-DAG maize seedlings were harvested and extracted with acetonitrile. In brief, samples were frozen in liquid nitrogen, ground to fine powder, and extracted with 0.5 mL 80% acetonitrile at -20^oC for 12 h. After centrifugation (10,000g, 4^oC, 20 min), the supernatants were collected and evaporated under mild nitrogen stream at 35^oC, followed by re-dissolving in 100 µL acetonitrile for UHPLC-MS/MS analysis. Internal standards were added to each sample, and the phytohormones were measured using a standard curve. For the endogenous ACC contents measurement, 500-mg samples were frozen in liquid nitrogen, ground to fine powder, and extracted with 0.5 mL MeOH at -20^oC for 12 h. After centrifugation (10,000g, 4^oC, 20 min), the supernatants were collected and evaporated under mild nitrogen stream at 35^oC, followed by re-dissolving in 100 µL 50%MeOH for UHPLC-MS/MS analysis. The UHPLC − MS/MS detection was performed in negative ion mode. Ion source conditions were as follows: sheath gas flow, 35 Arb; auxiliary gas flow, 20 Arb; sweep gas flow, 8 Arb; collision gas, 1.5 mTorr; ion transfer tube temperature, 350°C; vaporizer temperature, 300°C; spray voltage, 3.0 kV. The [²H₂]-JA, [²H₄]-SA, and [²H₄]-ACC (Olo-mouc, Czech Republic) were added as internal standards for quantification. Data acquisition and analysis was performed using a Thermo Scientific Xcalibur 2.1 data system. Three replicates were collected for each sample and the endogenous concentration of phytohormone contents was expressed as ng g^− 1 FW.

Lignin analysis. Seedlings were grown for 5 days with a paper-rolling method and cultured under the growth condition described. Then the seedlings with similar primary root length were chosen for ISX treatment. The root tips (~ 3cm) of the 5-DAG seedlings were immerged into water contained 1mM ISX and cultured under the same growth condition for 10 h, indicated as ISX (+); the root tips that were immerged into water added the same amount of DMSO as that of the added ISX were used as control, ISX (-). Lignification in seedling roots (n > 10) was analyzed 10 h after start of treatment. Lignified regions were detected with phloroglucinol-HCl as described³¹.

Luciferase complementation image (LCI) assay. For LCI assay, the coding sequence of ZmDRR206 was cloned into JW772 (C-terminal half of luciferase, cLUC) to produce cLUC-DRR206; and partial cDNA of ZmCesA10 described above and cDNA of ZmDIN1, ZmRin1 was cloned into JW771 (N-terminal half of luciferase, nLUC) to produce CesA10p-nLUC, DIN1-nLUC, RIN1-nLUC; using ClonExpress II One Step Cloning Kit (Vazyme Biotech) (primers were listed in Supplementary Table 2). These constructs were transformed into A. tumefaciens (strain GV3101), after which, the Agrobacterium cells were cultured to OD600 = 0.8, pelleted, and suspended in a buffer (10 mM methylester sulfonate, 10 mM MgCl₂, and 150 mM acetosyringone, pH 5.7). Equal amounts of OD600-normalized Agrobacterium cultures for cLUC and nLUC constructs were mixed to a final concentration of OD600 = 1.0. Transient expression in N. benthamiana leaf tissues was achieved by Agrobacterium infiltration. The suspended cells containing the above constructs were incubated at room temperature for ~ 3 h and infiltrated into 5-week-old Nicotiana benthamiana leaves in different combinations using a needleless syringe. The infiltrated plants were placed at 28°C for 48 h and then injected with 1 mM luciferin (Beetle luciferin, Promega) at the initial injection site, and then the fluorescence signal was imaged using a Tanon-5200 imaging system. These experiments were independently repeated three times and each combination was infiltrated with multiple leaves at each time.

Yeast two-hybrid assay. The DUALmembrane pairwise interaction assay in yeast was used to investigate the interaction between ZmDRR206 and ZmCesA10, ZmDIN1, ZmRIN1 according to the DUALmembrane User Manual (www.dualsystems.com). ZmDRR206 coding sequence without the N-terminal signal peptide was subcloned into pBT3-SUC DUALmembrane bait vector using a ClonExpress II One Step Cloning Kit (Vazyme Biotech), to generate pBT3-SUC-DRR construct, in which ZmDRR206 was fused to the C-terminal half of ubiquitin (Cub) and the artificial transcription factor LexA-VP16. And the coding sequence of ZmDIN1, ZmRIN1 and partial cDNA of ZmCesA10 was subcloned into sfiI digested pPR3-N (the pPR3-N DUALmembrane prey vector)) to generate pPR3-N-RIN1, pPR3-N-DIN1 and pPR3-N-CesA10p constructs, that is, these preys were fused to the mutated N-terminal half of ubiquitin (NubG) with primers listed in Supplementary Table 2. Plasmids pBT3-SUC-DRR was co-transformed into NMY51 yeast cell with pPR3-N-CesA10p, or pPR3-N-DIN1 or pPR3-N-RIN1, respectively. The combination of these prey constructs with pBT3-SUC empty, as well as the combination between pBT3-SUC-DRR and pPR3-N empty, was used as negative controls. Subsequently, the interaction was determined by spotting the resulting transformants onto synthetically defined (SD) /-Leu/-Trp and SD/-Ade/-His/-Leu/-Trp selection plates following the manufacturer’s protocols (www.dualsystems.com). NMY51 displays a pink color in the absence of a protein-protein interaction, similar to the yeast strains carrying an ade2 mutation; when NMY51 cells expressing an interacting protein pair will activate reporter gene to induce the cells to display a very faint pink to white color, depending on the strength of the interaction. ZmDRR206-antibody production and immunoblot analysis. To develop the ZmDRR206-specific antibody, the PSAVLAADDDGTTC peptide was chemically synthesized and used as antigen to immunize New Zealand white rabbits in Hangzhou Huaan Biotechnology (http://huabio.bioon.com.cn/). For western blotting, total proteins were extracted from 6-DAG seedlings with protein extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.2% NP-40, 5 mM DTT and 1×protease inhibitor cocktail). The membrane proteins were extracted from 6-DAG seedlings with Membrane Protein Extraction Kit (Sangon Biotech, C500049). 10 × SDS loading buffer was added into the protein samples, and the mixtures were incubated at 100°C for 5 min. Proteins were resolved by SDS–PAGE and then transferred to a PVDF membrane. The membrane was then incubated with primary and secondary antibodies and visualized using ECL Plus reagent.

Co-IP assay. To determine the ZmCES10-ZmDRR206 interaction in vivo, Co-IP assays were performed as previously described⁷⁴. In brief, Nicotiana benthamiana leaves coexpressing ZmDRR-GFP/GFP (control) and ZmCES10p-Myc were homogenized in immunoprecipitation buffer and centrifuged at 20,000 g for 20min at 4°C twice. GFP or GFP-tagged proteins were immunoprecipitated with GFP-Trap Magnetic Agarose beads (MBL, #D153-11). For protein blots, GFP and Myc fusion proteins were probed with anti-GFP (ABclonal, #AE012) and anti-Myc (ABclonal, #AE010) antibodies, respectively.

Statistical analysis. The statistical analysis was conducted using Student’s t-test between the WT and DRR-OE seedlings to determine statistical significance. The statistical significance was accepted at P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) for various growth or stress parameters. All gene expression analysis and statistical experiments were performed at least three times with independent samples. The data was expressed as the mean values ± SD of three replicates.

Acknowledgements

This work was supported by the Ministry of Agriculture and Rural Affairs of the People’s Republic of China (Grant No. 2018ZX0800917B) and grant from Yunnan Provincial Science and Technology Department (202005AF150026).

Competing interests

The authors declare that they have no competing interest associated with this work.

Author contributions

J. Ye, T. Zhong, and S. Deng performed the research; Y Li and X. Fan managed maize growth and inoculation in the field. M. Xu supervised this project; J. Ye designed the research, analyzed the data, wrote and revised the manuscript.

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ZmDRR206 involves in maintaining cell wall integrity during maize seedling growth and interaction with the environment

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