ACL1-HD-Zip IV complex revealed common mechanism in rice response to brown planthopper and drought

doi:10.21203/rs.3.rs-3811390/v1

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ACL1-HD-Zip IV complex revealed common mechanism in rice response to brown planthopper and drought

https://doi.org/10.21203/rs.3.rs-3811390/v1

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published 16 Sep, 2024

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Brown planthopper (BPH) is the most notorious insect pest to rice. Drought is the most commonly occurring global adversity. BPH infestation caused adaxially-rolled leaves and shrunk bulliform cells similar to drought. The bulliform-cell characteristic gene, ACL1, negatively regulated BPH resistance and drought tolerance, with decreased cuticular wax in ACL1-D, which resulted in quicker water losing. ACL1 was specifically expressed in epidermis. TurboID system and various biochemical assays revealed that ACL1 interacted with the epidermal-characteristic HD-Zip IV ROCs. ROC4 and ROC5 positively regulated BPH resistance and drought tolerance through modulating cuticular wax and bulliform cells respectively. Overexpression of ROC4 and ROC5 both rescued ACL1-D in various related phenotypes simultaneously. Moreover, ACL1 competed with ROC4 and ROC5 in homo-dimerization and hetero-dimerization. Altogether, we illustrated that ACL1-ROCs complex synergistically mediate drought tolerance and BPH resistance through regulating cuticular wax and bulliform cells in rice, a new mechanism which might facilitate BPH resistance breeding.

Biological sciences/Plant sciences/Plant molecular biology

Biological sciences/Plant sciences/Plant signalling

BPH

drought tolerance

ACL1

ROC

cuticular wax

Plants are constantly challenged by different kinds of biotic and abiotic factors simultaneously. Brown planthopper (BPH) (Nilaparvata lugens Stål) is the most notorious insect pest specific to rice, causing a yield lose toping other biotic stresses. In comparison with chemical insecticides and insect predators, endogenous genes render rice plant with long-lasting resistance, provide environmental-friendly ways for BPH control. Accordingly, 17 BPH resistance genes are cloned through till now (1–11). Elucidation of the underlying mechanism revealed that the signaling transduction is much similar to that of pathogen resistance (12, 13). BPH3 is a rice pattern recognition receptors (PRR) to mediate pattern-triggered immunity (PTI) response to BPH (4). While Bph14 and Bph26 encode NBS-LRR family proteins, which recognize effectors from BPH and induce effector-triggered immunity (ETI) (1, 3). Recent elucidation of BPH14 effector further verified the conservation of resistance mechanism against BPH and pathogens (14). However, rice plants might also evolved some distinctive defending mechanisms to cope with BPH, based on its specific infestation characteristics and damaging feature.

The typical symptom of BPH damage is withering of the plants till death, well known as “hopperburn”, indicating tight associated with water loss. Consistently, BPHs assemble at the base of rice stems and suck juice directly from the phloem, blocking sucking through deposition of callose (b-1, 3 glucan polymer) in the phloem cells could render BPH resistance (1, 15). Generally, the open/close status and density of the stomatal apparatus are the first and foremost factors that influence water-losing rate. Transpiration account for 90% total water loss under normal growth condition. But upon dehydration, the stomatal apparatus close, the cuticle layers controlling non-stomatal water-loss act. Meanwhile, cuticle layers constitute the first natural barrier to biotic stresses (16). Cuticular waxes are a mixture of hydrophobic very-long-chain fatty acids (VLCFA) and their derivatives accumulated in the plant cuticle (17). Drought can induce wax accumulation and even composition changes, and increased wax content on the leaf surface can improve drought tolerance (18–20). Genes influencing wax content in rice, such as Drought induced Wax Accumulation 1 (DWA1), Wax synthesis Regulatory 1 (OsWR1), OsGL1-6 and Drought Hypersensitive (DH) all influence water-losing rate and drought tolerance (20–24). Also, genes involved in VLCFA biosynthesis was up-regulated in small BPH (SBPH)-resistant rice plants (25), indicating possible connection between wax content and BPH resistance.

Drought is the most commonly occurring abiotic adversity that not only influences plant growth and development, but also reduces crop production and deteriorates global ecological environment (26). Rice provide food for more than one-half of world population, but its cultivation consumes a vast amount of water, making it even more vulnerable to drought throughout the growing season (27). Therefore, novel strategies for improving rice productivity in limited available water are critical for next-generation agriculture, which calls for study on the underlying mechanism of drought tolerance.

In rice, moderate leaf-rolling is helpful to form a well-proportioned leaf area for photosynthesis (28). Moreover, it might be tightly associated with drought tolerance (29). Leaf-rolling is the most obvious and quick response to drought, which is caused by the rapidly increased transpiration that could not be paced by water absorption. Bulliform cells are highly specialized epidermal parenchyma cells on the adaxial side of leaf surface in graminaceous plants that are highly sensitive to drought since they lose water immediately upon drought stress, and then turn relaxed and reduced in size so as to let the leaf roll inward to reduce further transpiration. Whether this kind of bulliform cell-associated leaf-rolling is related to BPH resistance is quite intriguing. Quite a few genes regulating bulliform cells are identified in rice, over expression of abaxially curled leaf 1 (ACL1) gene and zinc finger homeodomain class homeobox (OsZHD1) gene enlarges and increases the number of bulliform cells, and results in abaxially rolled leaves (30, 31). ROC5, ROC8, and LC2 negatively regulate development of bulliform cells, with their respective mutations enlarging bulliform cells (32–34). Meanwhile, variation in some genes causes adaxially rolled leaves due to reduced bulliform cells, such as OsLBD3-7, OsAGO7, NRL1, RL14, OsHB4 (35–39).

Therefore, BPH resistance might be tightly associated with drought tolerance, while the underlying mechanism remains largely unclear. Here we found that bulliform cells shrank similarly in response to BPH infestation and drought. Accordingly, the bulliform-cell-characteristic ACL1 gene negatively regulated BPH resistance and drought tolerance, with tremendously decreased wax content in the ACL1-D mutant. TurboID system identified several ROC proteins, and various biochemical assays confirmed the interaction between ACL1 and the ROC family. ROC5 and ROC4 positively regulated BPH resistance and drought tolerance. Specifically, ROC4 positively regulated wax content, and ROC5 positively regulated leaf-rolling. Both ROC5 and ROC4 could complement ACL1-D as to the BPH resistance, drought tolerance, wax content and leaf-rolling characters. Moreover, ACL1 compete with ROC4 and ROC5 in homo-dimerization and hetero-dimerization. Thus, we revealed that ACL1 synergistically regulate BPH resistance and drought tolerance in rice through repressing several ROC proteins.

Similar cytological response of rice plants to drought and BPH infestation

The typical symptom of rice plants under BPH infestation is the inward rolling of the leaves, followed by withering of the whole plants. Leaf-rolling is also a general response of plants to drought. In normal growth, the wild type (WT) rice plant ZH11 has flat leaves (Fig. S1A). Upon BPH infestation for 6 days, ZH11 leaves rolled inward (adaxially) (Fig. S1B). Similarly, the leaves rolled adaxially upon drought (Fig. S1C). Detailed observation revealed that the bulliform cells, whether adjacent to the main vein (Fig. 1A, black brackets), or adjacent to the secondary vein (Fig. 1A, red brackets), were all obviously shrunk compared with those under normal growth (Fig. 1A). Thus, the rice plants responded to drought and BPH infestation similarly, the external withering of the plants leaves were initiated by the shrinking of bulliform cells at the cellular level.

ACL1 negatively regulated drought tolerance and BPH resistance

ACL1 specifies the development of bulliform cells (30). To examine the tissue localization of ACL1 protein, we constructed pACL1:GUS plants and GUS signals was detected in almost all tissues including the seedling, root, leaf sheath, leaf, culm and spikelet (Fig. S2). Specifically, in the cross section of the root hair zone, the GUS signal was uniformly distributed (Fig. 1B), while in the root tip, it was much more concentrated and formed an “ACL1-GUS ring” of the epidermal cells (Fig. 1C). Thus, ACL1 is an epidermal protein.

Various genetic materials of ACL1 gene were constructed for genetic verification. The previous mutant with ACL1 up-regulation (Fig. S3A) and abaxially-rolled leaves (Figs. S3B, C) (32) was re-named as ACL1-D. ACL1 over expression plants fused with MYC, ACL1-MYC, was constructed in NIP background (Figs. S3D, E), and the leaves were abaxially rolled (Figs. S3F, G). Also, the Arabidopsis AtACL1 gene was over expressed in rice (Fig. S3H), and the resulted AtACL1OE plants showed abaxially-rolled leaves (Figs. S3I, J). Thus, the function of ACL1 gene in abaxial leaf curling was conserved between rice and Arabidopsis. Meanwhile, we used Crisper-CAS9 technology and constructed ACL1 edited ACL1KO plant with a deletion of two nucleotides (nt) (Fig. S4B). In rice, there is another homolog of ACL1 (Fig. S4A), ACL2. We made double edited ACLDKO plants of both genes, which were respectively edited in ACLDKO-15 (Fig. S4C) and ACLDKO-23 (Fig. S4D) lines. ACL1KO and ACLDKO plants all showed flat leaves (Figs. S4E-H).

Two methods were used for drought tolerance test, direct water cut-off and 20% PEG6000 treatment to simulate drought stress. In direct water cut-off, more ACL1-D plants died than WT (Fig. S5), with a lower survival rate (Fig. 1D). While less ACL1KO plants died than WT (Fig. S5), with a higher survival rate (Fig. 1D). Also, ACLDKO-15 and ACLDKO-23 plants withered later than WT (Fig. S5), both with higher survival rates (Fig. 1D). Under 20% PEG6000 treatment, similar results were got (Fig. S6). Together, these data proved that ACL1 negatively regulated drought tolerance. Furthermore, we tested the response of the AtACL1OE and ACL1-MYC plants to drought, whether in water cut-off assay or in 20% PEG6000 treatment, AtACL1OE and ACL1-MYC plants withered earlier than WT, with lower survival rates (Figs. S7, S8). Altogether, over expression of ACL1 in different genetic backgrounds, and over expression of Arabidopsis AtACL1 in rice were all sensitive to drought; further verifying the negative regulation of ACL1 in drought response, and conservation of ACL1 between rice and Arabidopsis.

BPH attack causes water losing, now that ACL1 negatively regulated drought tolerance, we wonder if it regulates BPH resistance. Individual test and small population test were used. In both kinds of assays, ACL1-D plants died earlier than ZH11 (Figs. 1E, F), with a lower survival rate (Fig. 1G), indicating a susceptible character of ACL1-D to BPH. When the resistant variety RHT, the susceptible variety TN1, ACL1-D and ZH11 were compared in parallel, the susceptible character of ACL1-D was similar to TN1, when most ACL1-D and TN1 died, ZH11 turned yellow, while RHT plants were still green (Fig. 1H). Meanwhile, AtACL1OE was susceptible to BPH in both kinds of tests (Figs. S9A, B), with a lower survival rate (Fig. S9C). Also, in comparison with RHT and TN1, the susceptible level of AtACL1OE plants was similar to that of TN1 (Fig. S9D). Also, the ACL1-MYC plants were more susceptible to BPH than NIP in both kinds of tests (Figs. S9E, F), with a lower survival rate (Fig. S9G). Moreover, the ACL1KO plants died later than the WT plants (Figs. 1I, J), with a higher survival rate (Fig. 1K), indicating a resistant character. Similarly, ACLDKO-15 and ACLDKO-23 plants died later than WT in both kinds of tests (Figs. 1L, M, O), with higher survival rates (Figs. 1N, P). These results collectively indicated that ACL1 negatively regulated BPH resistance.

Three kinds of physiological mechanism were usually used by plants to invert herbivory insects, antibiosis, antixenosis and tolerance (40). To make out which kind of mechanism was used by ACL1-D and ACL1KO plants against BPH, BPH weight-gain test was carried out, but no difference was observed (Fig. S10A). Neither, the choice test revealed no significant differences between the numbers of BPH settled on the ACL1-D, ACL1KO and ZH11 plants after infestation (Fig. S10B). Further, in the tolerance test, it was revealed that the functional plant loss (FPL) index of the ACL1-D plants was lower than that of ZH11, while that of ACL1KO plants was higher (Fig. 1Q). And both the plant dry weight loss and the plant dry weight loss to BPH dry weight (PDWL) of the ACL1-D plants were higher than that of ZH11, while those of ACL1-D plants were lower (Figs. 1R-S). These results collectively indicated that neither antibiosis nor antixenosis was used by ACL1-D and ACL1KO plants against BPH, but tolerance mechanism accounted.

Wax content was changed in the ACL1-D and ACL1KO plants

To explore the underlying mechanism of ACL1 gene, we carried out an mRNA-seq assay of the ACL1-D and ZH11 plants. In the KEGG-pathway analysis, the fatty acid elongation pathway and the lipid metabolism pathway were obviously enriched in the ACL1-D/ZH11 pairs (Fig. S11), indicating possible influence on wax content. We therefore checked the leaf surface of ACL1-D, ACL1KO and ZH11 plants using scanning electron microscope (SEM), and found there was less wax on the ACL1-D leaf surface, while more on the ACL1KO leaf surface compared with that on ZH11 (Figs. 2A-C). Also, on the leaf sheath surface, the wax was much denser in the ACLDKO plants while even sparser in the ACL1-D plants (Figs. 2D-F). Further quantification in gas chromatography mass spectrometry (GC-MS) assay revealed that in the ACL1-D plants, the waxes, especially those C22-C30 long-chain-fatty acids were obviously lowered, while those in the ACL1KO plants were increased (Fig. 2G). Also, on the leaf surface of the ACLDKO-15 and ACLDKO-23 plants, there was more wax than on the ZH11 leaf surface (Figs. S12A-C), and the wax content was higher in both ACLDKO lines checked by GC-MS (Fig. S12D).

Since wax content might influence the permeability of the leaf surface, we further detected the water loss rate in ACL1-D, ACL1KO and ZH11 plants, it was revealed that the dry weight of the ACL1-D plants decreased much quicker than that of the ZH11 plants, while that of the ACL1KO plants was much slower (Fig. 2H). Meanwhile, we detected the chlorophyll leaching rate, although the ACL1KO plants did not show any difference with ZH11, the ACL1-D plants showed much higher rate of chlorophyll leaching (Fig. 2I). Accordingly, ACL1 influenced the wax content on the plant surface, which influenced water permeability.

ACL1 interacted with almost all ROC proteins in rice

Being a small peptide, ACL1 might function through forming complex with other proteins. So that we searched the possible interacting proteins of ACL1 using the TurboID system (41, 42). We first constructed the Turbo-ACL1 and Turbo transgenic plants. As anticipated, the Turbo-ACL1 plants showed ACL1 over expression (Fig. S13A), with the fusion Turbo-ACL1 protein readily detectable (Fig. S13B), and the abaxially rolled leaves were obvious (Figs. S13C, D), but the Turbo plants did not (Fig. S13E), indicating successful over expression of Turbo-ACL1 protein, and the fused Turbo protein did not disturb with the function of ACL1 protein.

Then we used the Turbo-ACL1 and Turbo plants to carry out immuno-precipitation followed by protein liquid chromatography mass spectrometry (LC-MS) assay. The precipitated proteins identified were summarized in Table S1. Among them, there were ROC2 and ROC5 proteins belonging to the homeodomain leucine zipper IV (HD-Zip IV) transcriptional factor (TF) family, most of which are epidermal or sub-epidermal cells specific (43), considering the epidermal expression character of ACL1 (Fig. 1C), we further verified the interaction between ACL1 and these ROC proteins. There are totally nine genes encoding ROC proteins in rice (Fig. S14A) (43). While ROC9 was hardly detectable, others showed a relative high expression in the leaves (Fig. S14B). We cloned the ROC1-8 genes, and carried out yeast two hybrid (Y2H) (Fig. S15), bimolecular fluorescence complementation (BIFC) (Fig. S16), luciferase complementation imaging (LCI) (Fig. 3A) and Co-immunoprecipitation (Co-IP) assays (Fig. 3B), all of which confirmed the interaction between ACL1 and each ROC1-8 proteins respectively.

ACL1 compete with ROC4-ROC4 and ROC5-ROC5 homodimers, and ROC4/5 heterodimer

Plant HD-Zip IV family members function via homodimers and heterodimers (33). We therein checked ROC4 and ROC5. When Nluc-ROC4 was co-expressed with ROC4-Cluc in tobacco leaves, the LUC signal was strong in the nucleus (Figs. 3C, S17A), indicating ROC4 could form homodimer. However, when ACL1 was added, the interaction between ROC4-Cluc and Nluc-ROC4 became weaker as indicated by the decreased LUC signal (Figs. 3C, D). Similarly, when Nluc-ROC4 was co-expressed with ROC5-Cluc, the LUC signal was strong in the nucleus (Figs. 3E, S17B), indicating ROC4 could form heterodimer with ROC5. However, in the presence of ACL1, the strength of the inflorescence signal was decreased (Figs. 3E, F). Meanwhile, when Nluc-ROC5 was co-expressed with ROC5-Cluc, the LUC signal was strong in the nucleus (Figs. 3G, S17C), indicating ROC5 could form homodimer. However, in the presence of ACL1, the strength of inflorescence signal was decreased (Figs. 3G, H). These assays indicated that ACL1 interfered with the formation of ROC4-ROC4, ROC4-ROC5 and ROC5-ROC5 homodimers and heterodimers.

We further carried out competitive Co-IP. Consistently, ROC4-HA was co-immuno-precipitated by ROC4-Flag with Flag-Trap (Fig. 3I), when ACL1-GFP, but not GFP itself, was co-expressed with ROC4-HA and ROC4-Flag, the amount of ROC4-HA co-immuno-precipitated by ROC4-Flag decreased tremendously (Fig. 3I), indicating that homo-dimerization of ROC4 decreases by ACL1. Similarly, ROC4-HA was co-immuno-precipitated by ROC5-Flag with Flag-Trap, when ACL1-GFP, but not GFP itself, was co-expressed with ROC4-HA and ROC5-Flag, co-immunoprecipitation of ROC4-HA and ROC5-Flag decreased (Fig. 3J). Meanwhile, ROC5-HA was co-immuno-precipitated by ROC5-Flag with Flag-Trap, when ACL1-GFP, but not GFP itself, was co-expressed with ROC5-HA and ROC5-Flag, co-immunoprecipitation of ROC5-HA and ROC5-Flag decreased (Fig. 3K).

ROC4 positively regulated drought tolerance, BPH resistance and wax content

Several ROC genes have been reported to regulate leaf-rolling in rice, such as ROC5 (32) and ROC8 (33, 44), specifically, roc5 and roc8 mutants showed enlarged and increased bulliform cells similar to ACL1-D. ROC4 has been reported to mediate drought tolerance through regulating wax synthesis (21). Now that ACL1 interacts with ROC1-8 proteins, we analyzed the genetic function of some of these ROC proteins in drought tolerance and BPH resistance.

We constructed the ROC4KO plants and ROC4OE plants and selected two lines each for further function analysis. In the ROC4OE plants, ROC4 was up-regulated (Fig. S18A), and ROC4 was successfully edited in the ROC4KO-1 and ROC4KO-2 plants (Fig. S18B). SEM assay revealed that in the ROC4KO-1 and ROC4KO-2 plants, wax was sparser in the leaves, while in the ROC4OE-20 and ROC4OE-24 plants, it was much denser (Fig. 4A). GC-MS assay further verified that the wax content was lower in the ROC4KO-1 leaves, while higher in the ROC4OE-24 leaves (Fig. 4B). We further carried out drought tolerance and BPH resistance assays. When treated with 20% PEG6000, the ROC4KO-1 and ROC4KO-2 plants survived worse than ZH11 (Fig. 4C), with lower survival rates (Fig. 4D). While the ROC4OE-20 and ROC4OE-24 plants survived better than ZH11 (Fig. 4E), with higher survival rates (Fig. 4F). Furthermore, in direct water cut-off test, the sensitive character of both lines of ROC4KO plants and the tolerant character of both lines of ROC4OE plants were further verified (Fig. S19). Meanwhile, in both individual test and small population test for BPH resistance detection, the ROC4KO-1 and ROC4KO-2 lines died earlier than WT (Figs. 4G, H, J), with the survival rates of both lines lower than that of WT (Figs. 4I, K). While the ROC4OE-20 and ROC4OE-24 lines died later than WT (Figs. 4L, M, O), with the survival rates of both lines higher than that of WT (Figs. 4N, P). Therefore, ROC4 positively regulates drought tolerance, BPH resistance and wax content simultaneously in rice.

ROC5 was needed for drought tolerance and BPH resistance

Similarly, we constructed the ROC5KO plants (Fig. S20A), which also showed abaxially-rolled leaves (Fig. S20B) similar to the roc5 mutant (32). In drought tolerance test, the ROC5KO plants were much sensitive to drought than the WT in direct water cut-off (Fig. 5A), with a lower survival rate (Fig. 5B). When treated with 20% PEG6000, similar results were got (Figs. 5C, D). Meanwhile, in both kinds of BPH resistance tests, the ROC5KO plants died earlier than ZH11 (Figs. 5E, F), with a lower survival rate (Fig. 5G). Thus, ROC5KO plants was not only sensitive to drought but also vulnerable to BPH infestation. Similar results were got using roc5 mutant and WT NIP. roc5 mutant is susceptible to BPH whether in individual test (Fig. S21A) or small population test (Fig. S21B), with a lower survival rate (Fig. S21C). Meanwhile, whether in direct water cut-off (Figs. S21D, E) or 20% PEG6000 treatment (Figs. S21F, G), the roc5 mutant was more sensitive to drought. Thus, ROC5 gene was needed for both drought tolerance and BPH resistance.

Over expression of ROC4 and ROC5 in ACL1-D recovered the drought sensitive, BPH susceptible, wax content and leaf-rolling phenotypes

Further, we investigated the genetic interaction between ACL1 and these ROC genes. First, we over expressed ROC4 in the ACL1-D plants and got ROC4OE/ACL1-D (abbreviated as 4OE/ACL1-D in figures) plants. Expression of both ACL1 and ROC4 in ROC4OE/ACL1-D plants was respectively higher than that in ZH11 (Fig. S22A). Next, in direct water cut-off assay, the drought sensitive character of the ACL1-D plants was recovered in the ROC4OE/ACL1-D plants (Fig. 5H), with the survival rate of ROC4OE/ACL1-D plants similar to that of ZH11, both much higher than that of ACL1-D (Fig. 5I). In BPH resistance assays, the ACL1-D plants died when both the ZH11 and ROC4OE/ACL1-D plants were still alive in individual test (Fig. 5J) and small population test (Fig. 5K), and the survival rates of ZH11 and ROC4OE/ACL1-D plants were similar, both much higher than that of ACL1-D plants (Fig. 5L). Therefore, over expression of ROC4 recovered the drought sensitive and BPH vulnerable characters of ACL1-D plants. We further carried out SEM assay to see if his recover is associated with wax content, it was revealed that the wax status on the leaf surface of ROC4OE/ACL1-D plants is similar to that on ZH11 leaf surface, which is much denser than that on the ACL1-D leaf surface (Fig. 5R). Furthermore, GC-MS assay revealed that the wax content in ROC4OE/ACL1-D plants showed no difference to that in ZH11 (Fig. S23A). Therefore, the less wax in ACL1-D plants was also recovered by ROC4 over expression.

Also, we over expressed ROC5 in the ACL1-D plants and got ROC5OE/ACL1-D (abbreviated as 5OE/ACL1-D in figures) plants. Expression of both ACL1 and ROC5 in ROC5OE/ACL1-D plants was respectively higher than that in ZH11 (Fig. S22B). In direct water cut-off assay, the drought sensitive character of the ACL1-D plant was recovered in the ROC5OE/ACL1-D plants (Fig. 5M), with the survival rate of ROC5OE/ACL1-D plants similar to that of ZH11, but both much higher than that of ACL1-D (Fig. 5N). In BPH resistance assays, the ACL1-D plants died when both the ZH11 and ROC5OE/ACL1-D plants were still alive in both individual test (Fig. 5O) and small population test (Fig. 5P), and the survival rates of ZH11 and ROC5OE/ACL1-D plants were similar, both much higher than that of ACL1-D plants (Fig. 5Q). Therefore, over expression of ROC5 recovered the drought sensitive and BPH vulnerable characters of ACL1-D plant. We further carried out SEM analysis and revealed that the wax status on the leaf surface of ROC5OE/ACL1-D plants is similar to that on ZH11 leaf surface, which is much denser than that on the ACL1-D leaf surface (Fig. 5R). Furthermore, GC-MS assay revealed that the wax content in ROC5OE/ACL1-D plants showed no difference to that in ZH11 (Fig. S23B). Therefore, the less wax in ACL1-D plants was also recovered by ROC5 over expression. Although, in the ROC5OE plants, the wax content was similar to that in ZH11 (Figs. S24A-C, Fig. S24E), neither did the wax content in ROC5KO showed difference to that in ZH11 (Figs. S24A, D, E). Although ROC5 gene was up-regulated (Fig. S20C) and the leaf was adaxially rolled, as contrary to that of ROC5KO plants, which was abaxially rolled (Fig. S20B).

We further detected the leaf-rolling character of the ROC4OE/ACL1-D and ROC5OE/ACL1-D plants. Although the ROC4OE leaf did not rolled adaxially (Figs. 5S, U), the ROC4OE/ACL1-D plant leaf was flat as ZH11, recovering the abaxially rolled leaf character of ACL1-D (Figs. 5S, T, V). And the ROC5OE/ACL1-D leaf was also flat (Figs. 5S, X), intermediate of the adaxially rolled ROC5OE leaf (Fig. 5W) and the abaxially rolled ACL1-D leaf (Fig. 5T). Thus, the rolled leaf character of ACL1-D plant was recovered by respective over expression of ROC4 and ROC5.

To sum up, respective over expression of ROC4 and ROC5 in ACL1-D recovered the drought sensitive, BPH susceptible, low wax content, and the leaf-rolling phenotypes simultaneously.

ACL1-ROCs complexes synergistically regulated drought and BPH resistance

Now that ACL1 inhibited the function of the ROCs, we further checked if these repressor complexes recruit TOPLESS. In the TurboID system, the three homologies of TOPLESS in rice, TPR1, TPR2, TPR3 were all filtered as neighboring proteins of ACL1 (Table S1, Fig. S25A). Meanwhile, there was an EAR motif in the ACL1 protein which indicated possible TOPLESS interaction (Fig. S25B). We next used LCI system to investigate the interaction between ACL1 and these TOPLESS proteins and revealed that the combination of TPR3 and ACL1 showed strong fluorescence signal (Fig. S25C). Thus ACL1 might recruit TPR3 to form repressive complex.

Altogether, the mechanism of ACL1-ROCs complexes synergistically regulating drought and BPH resistance was revealed. When there is less ACL1, more ROCs are released from the ACL1-ROCs complexes to form homodimers or heterodimers to regulate downstream genes. For example, ROC4 regulates wax synthesis genes to promote wax at the leaf and sheath surface, and ROC5 regulates genes to inhibit the over-development of bulliform cells in the adaxial epidermis, thus comprehensively promoted drought tolerance and BPH resistance (Fig. 6A). When ACL1 is over-produced, it competitively binds with ROC4 and ROC5 from their respective homodimers or heterodimers, and recruits TOPLESS to inhibit the downstream transcription on wax genes and bulliform cell related genes, thus results in less wax and enlarged and increased bulliform cells, which promoted the vulnerability to both drought and BPH (Fig. 6B).

Trichromes are specialized epidermis cells that deter insects. Similarly, bulliform cells are specialized epidermis cells specific to grass family, with the function and underlying mechanism in stress response unclear. In maize, bulliform cells have specialized water-permeable cuticle and shrank differentially during leaf dehydration (45). Here we showed that in rice upon drought and BPH infestation, the bulliform cells shrank (Fig. 1A), along with the leaf adaxially rolling (Fig. S1), moreover, the ACL1-D mutant with increased bulliform cells was more sensitive not only to drought (Figs. 1D, S5), but also to BPH infestation (Figs. 1E-G), with decreased cuticular wax content which might promote water permeation (Fig. 2). Furthermore, in both the ACL1KO and ACLDKO plants, the wax contents were increased (Figs. 2C, F, G; Fig. S12), and accordingly both ACL1KO and ACLDKO plants were more BPH resistant (Figs. 1I-P) and drought tolerant (Figs. 1D, S5, S6). Consistently, the ACL1 interacting protein ROC5 function to inhibit development of bulliform cells, in the ROC5KO plants, the bulliform cells were enlarged and increased and resulted in abaxially leaf-rolling (Fig. S20B), similar to ACL1-D, and also, the ROC5KO plants were drought sensitive (Figs. 5A-D) and BPH susceptible (Figs. 5E-G). Therefore, the bulliform cells takes the initiative to respond in stresses-induced leaf-rolling in rice, the parenchyma cell character together with the covering wax might contribute to this function.

The cuticle forms a hydrophobic waxy layer that covers plant organs and provides physical protection from biotic and abiotic stresses. Accumulation of cuticular waxes contributes to drought tolerance (46), and some TFs modulate drought tolerance through regulating wax content (19, 47). The wax was greatly decreased in ACL1-D plants (Figs. 2B, E, G), while increased in the ACL1KO plants (Figs. 2C, F, G), indicating possible involvement of wax in the ACL1-meditaed drought tolerance and BPH resistance. Consistently, the interacting ROC4 positively regulated wax content, with ROC4OE increased, while ROC4KO decreased wax content (Figs. 4A, B), as a result, ROC4OE plants were drought tolerant (Figs. 4E, F, S19 E-H) and BPH resistant (Figs. 4L-P), while ROC4KO plants were drought sensitive (Figs. 4C, D, S19A-D) and BPH susceptible (Figs. 4G-K). Therefore, ACL1-ROC4 might modulate BPH resistance and drought tolerance through regulating wax content, ROC4 directly regulates wax biosynthesis genes (21), and ACL1 modulates ROC4 though directing interaction at the protein level (Figs. 3A, B), which disturbed the formation of ROC4 homodimers (Figs. 3C, D, I), and/or heterodimer with other ROCs such as ROC5 (Figs. 3E, F, J).

ACL1 gene showed an epidermal expression character in the root (Fig. 1C). In accordance, over expression of ACL1 resulted in enlarged and increased bulliform cells which resulted in abaxially leaf-rolling (30) (Fig. S3C). The homologous genes of ACL1 in Arabidopsis, GIR1 and GIR2, regulate root hair formation, showing an epidermal character either (48). Meanwhile, over expression of GIR1 in rice resulted in abaxially rolled leaves (Fig. S3J), and enhanced drought sensitivity (Fig. S7) and BPH susceptibility (Figs. S9A-D), indicating the function conservation of ACL1 in Arabidopsis and rice not only in development, but also in physiology against biotic and abiotic stresses. Moreover, in Arabidopsis, GIR1 and GIR2 interact with GLABRA2 (48), a typical HD-Zip IV protein that suppresses root hair differentiation as well as initiates leaf trichome development (49, 50). Here we showed that ACL1 interacted with HD-Zip IV family ROC proteins, which show typical epidermal expression character and functioning (43). Moreover, the ACL1-ROC interaction was also conserved in tomato, with the Woolly (HD-Zip IV) and SlCycB2 (homologous to ACL1) pair regulating trichrome formation (51). Furthermore, NtCycB2 in Nicotiana tabacum negatively regulates trichome development and aphid resistance (52). Thus, the epidermal characteristic complex ACL1-ROCs might be conserved among plant species. HD-Zip IV drives the differentiation of epidermal cells (53), ACL1 might be a conserved co-factor of HD-Zip IV, the extent to which this ACL1-ROC complex function in plant kingdom deserves further research.

The insect feeding behavior (piercing-sucking versus chewing) is a decisive determinant of the plant's defense response (54). The interaction between rice and BPH are typical for piercing-sucking insects. Although increasing evidence indicated the similarity between the perceiving mechanism and the following responsive signaling transduction against BPH resistance and microbial pathogen (12, 13), more specific genetic and molecular mechanism based on particular feeding behavior might be neglected. Response caused by piercing-sucking insects is usually not associated with a strong wound response as those chewing insects, but might be tightly associated with a drought response due to the direct liquid sucked out from the phloem during feeding. In accordance with this hypothesis, we revealed that the ACL1-ROC5, ACL1-ROC4 pathways synergistically regulate drought tolerance and BPH resistance through regulating the development of bulliform cells and cuticular wax content respectively (Fig. 6). Tight cooperation might exist between ACL1-ROC4 and ACL1-ROC5 pairs, since ROC4 and ROC5 could interact, and ACL1 could compete with the ROC4-ROC5 heterodimer (Figs. 3E, F, G). If the mechanism of this epidermal characteristic ACL-ROCs complex is also applicable to other kinds of interaction between piercing-sucking insects and host plants still needs further study. If applicable, the wax content might be a conserved downstream factor mediating the resistance to insects, however, the grass family-specific bulliform cells might be adjusted to other kinds of specialized epidermal cells in other plant species, such as trichomes, that could deter insects.

All together, we discovered a kind of ACL1-ROCs complexes in rice that could synergistically regulate drought tolerance and BPH resistance through modulating two kinds of typical physical barriers in the plant surface, bulliform cells and cuticular wax (Fig. 6). With increased wax content and/or shrunk bulliform cells, the rice plants are more tolerance to drought and resistant to BPH (Fig. 6A), to the contrary, with increased bulliform cells and/or decreased wax content, the rice plants are more susceptible to BPH and more vulnerable to drought (Fig. 6B).

Plant species and growth conditions

The wild type (WT) rice plants were varieties ZH11 (Oryza sativa L. subsp. japonica cv. Zhonghua No.11, ZH11), NIP (Japonica. cv. Nippobare, NIP), RHT (Rathu Heenati) and TN1 (indica cv. Taichung Native 1, TN1). ZH11 and NIP were mainly used as host of genetic transformation or natural WT of mutants. RHT was a well-known BPH-resistant variety. TN1 was used to cultivate BPH. All rice plants were cultivated under field conditions in two experimental stations in Shanghai (30°N, 121°E) and Lingshui (Hainan Province, 18°N, 110°E), China. Rice seedlings were cultures in the phytotron in CAS Center for Excellence in Molecular Plant Sciences, with 30/24 ± 1℃ day/night temperature, 50–70% relative humidity and a light/dark period of 14 hours/10 hours.

Plasmid construction and plant transformation

For over expression of AtACL1, ROC4 and ROC5, full length cDNA of respective genes were amplified and cloned into p1301-35S-Nos vector through digestion by XbaI and KpnI.

For over expression of ROC4 and ROC5 in the ACL1-D background, full length cDNAs of them were respectively amplified and cloned into the pCAMBIA2301 vector through digestion by XbaI and KpnI.

For construction of pACL1:GUS, the 1.9 kb fragment upstream of the ATG start code of ACL1 was amplified and cloned into the PstⅠ and XbaⅠ sites of the p1300-GUS-Nos plasmid by homologous recombination using the Vazyme ClonExpress® Ultra One Step Cloning Kit.

For construction of knock-out plants, guider DNA was respectively synthesized and cloned in the pOs-sgRNA vector, and then transferred to the pH-Ubi-cas9-7 vector through LR reaction. Primers and gDNAs used were listed in Table S2.

For fusion with the Turbo, the ACL1 sequences were independently amplified and cloned into pCRTM8/GW/TOPO® and then transferred into Gateway-compatible 35S-YFP-miniTurbo vectors by LR reaction (60).

Plasmids were transformed into ZH11 or ACL1-D mutant through Agrobacterium-mediated genetic transformation in Biorun Biosciences Company.

Calculation of water loss efficiency

The water loss efficiency assay was performed as described (61). 4-week-old rice seedlings were cut from the base and weighed with a microbalance at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 h. The water loss efficiency was calculated according to the initial weight of the samples. Eight replicates were set in each group.

Chlorophyll leaching assay

Chlorophyll leaching assay was performed as described (62). 2 cm segments were removed from the middle part of the second leaf of 4-week-old seedling, and immediately soaked in 2 ml 80% ethanol, and put in the dark. At 1, 2, 3, 4, 5, 6 and 24 h time points, the absorption spectra at 647 and 664 nm wavelengths were measured by Thermo NANODROP ONE in the order of removal, and the chlorophyll content was equal to 7.93*A664 + 19.53*A647. The chlorophyll leaching rate at each time point was calculated according to the chlorophyll content of the samples at 24 h.

TurboID-mediated proximity labelling proteomics

Biotin treatment and protein extraction. 7-day-old Turbo-ACL1 seedlings were immersed in 50 µM biotin solution for 3 h, and quickly frozen in liquid nitrogen after rinse with ice water. Samples were grinded, and added pre-cooled Extraction buffer (50 mM Tris-HCL (pH 7.5), 150 mM NaCl, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 µg/mL leupeptin and 1× protease inhibitor cocktail), and centrifuged at 12000 rpm, 15min, 4℃ to collect the supernatant. Repeat the centrifuge and collection process once.

Protein affinity purification. Biotin was removed from the supernatant using Zeba™ Spin Desalting Columns (Thermo Fisher Scientific). To enrich biotinylated proteins, 30 µl Streptavidin Magnetic Beads (MedChem Express) were added into 2 ml centrifugation tubes, and washed twice with the pre-cooled extraction buffer, then the desalted protein extracts were added and incubated overnight on a 4℃ spinner. Then the magnetic beads were cleaned after magnetic separation according to Mair et al.(63). Specifically, used cold extraction buffer, cold 1 M KCl, cold 100 mM Na₂CO₃, 2 M Urea in 10 mM Tris pH 8 at room temperature once each, and cold extraction buffer without complete and PMSF twice to clean the magnetic beads. Then 5% magnetic beads were taken for detection by Streptavidin-HRP (Abcam).

Digestion of the beads. The magnetic beads were cleaned with 50 mM ammonium bicarbonate water 3 times. After magnetic separation, 200 µl 10 mM TCEP and 50 mM CAA prepared with 50 mM ammonium bicarbonate were added and incubated at 37℃ for 30 min to redox and alkylate the disulfide bonds. Then digested with trypsin (Promega) at 37°C overnight after rinse with 50 mM ammonium bicarbonate. The digests were desalted using PIERCE C18 SPIN COLUMNS (Thermo fisher), and then ran liquid chromatography mass spectrometry (LC-MS) detection.

Sequence Database Searching and Data Analysis

The MS data were analyzed using Thermo proteome Discoverer2.5.0.400 software, and searched against the Rice_Oryza sativa subsp japonica_UP000059680_39947. An initial search was set at a precursor mass window of 10 ppm, followed by an enzymatic cleavage rule of Trypsin and allowed maximal two missed cleavage sites and a mass tolerance of 0.02 Da for fragment ions. Carbamido methylation of cysteines was defined as fixed modification, while protein N-terminal acetylation and methionine oxidation were defined as variable modifications for database searching. The cutoff of global false discovery rate (FDR) for peptide and protein identification was set to 0.01.

Phylogenetic analysis

The protein sequence of all the genes were downloaded from National Center for Biotechnology Information databases (https://www.ncbi.nlm.nih.gov). Phylogenetic trees were generated using predicted full-length amino acid sequences by the maximum-likelihood method in MEGA7.0 with bootstrap mode and 500 replications.

Western blot

Western blot was performed according to the procedures described previously (64).

BPH resistance detection and measurements

The BPH population was originally obtained from rice fields in Shanghai, China, and maintained on susceptible rice variety TN1 in a climate-controlled room at 26 ± 2°C, 12h/12h light/dark cycle and 80% relative humidity.

Individual test assay was carried out at seedling stage using at least six replicates of each cultivar or line as previously described (8, 65). Each seedling about 5 weeks was infested with twelve second-instar BPH nymphs. Plant status were checked daily, and about 5–12 days later, the plants were scored as susceptible (dead) or resistant (alive) and photographed.

For small population assay, about 40 plants of tested lines and the WT were planted in the mud in a plate for one month till about third-leaf stage, and fed to BPH population in appropriately 10–15 first-instar nymphs per plant, and the plant status were surveyed daily. Plants were photographed and the survival rates was calculated based on data from at least three repeats.

For selection mechanism detection, each 2 germinated seeds of ACL1KO, ACL1-D and ZH11 were evenly planted in the mud in a small pot with 10 repeats. About 6 weeks later, about 400 3-instar nymphs of BPH were collected and placed in the middle of the pot to let them scatter randomly. Each pot was covered by a nylon mesh. The number of BPH on each plant was counted at 24 h, 48 h and 72 h after infestation.

Each four plants of ACL1KO, ACL1-D and ZH11 were planted in the mud in small pots with 10 repeats. About 6 weeks later, 200 2-instar nymphs of BPH were weighed to get an initial weight and then put to the plants after starvation for 2 hours. Each line was arranged with 5 replicates, and another 5 replicates without feeding were simultaneously set up. After 7 days, the BPH were collected and weighed to calculate the weight gain. Meanwhile, the above-ground portions of both infested and uninfected plants were collected, dried and weighed. Tolerance-related indicators refer to Sarao et al. (66). Plant dry weight loss (mg) = dry weight of non-infested plant-infested plant; Functional plant loss index (FPL, %) = (1- dry weight of infested plant/dry weight of uninfected plant) ×100; Plant dry weight loss to BPH dry weight produced (PDWL) = Plant dry weight loss/BPH weight gain.

Anatomical analysis

Anatomical sections of the GUS stained tissues were carried out as described (55).

For manual sections, freshly collected flag leaves were manually cut into about 0.5 mm slices in the middle part as quickly as possible, and pictures were taken under NIKON D7000.

Histochemical GUS staining

GUS staining was carried out as previously described (56). Photographs were taken under a stereo Leica M205FA.

Drought tolerance detection methods

For direct water cut-off, germinated seeds of the tested and control plants (about 40 plants for each line) were planted in parallel in the paddy soil in a 13×18 cm plastic box, grown in the phytotron for 4 weeks, then stopped watering until the plant withered (at about 5-11days), and re-watered again for 3–7 days to check the recovery status. The survival rates were calculated based on data from at least three repeats.

For 20% PEG6000 treatment, germinated seeds were planted in plastic plate in the rice nutrient solution (Yoshida) (changed every 2 days) in the phytotron for 3 weeks. Treatment was carried out using 20% (w/v) PEG6000 solution solved in the rice nutrient solution until the plants were wilted (at about 6–20 days), and then replaced with the rice nutrient solution again (re-watering) for 3–7 days to check the recovery status. The survival rates was calculated based on data from at least three repeats.

Scanning electron microscope (SEM) assay of the leaf/sheath surface

The flag leaves or the leaf sheaths was removed and quickly frozen in liquid nitrogen and then freeze-dried. The samples were observed by high resolution field emission SEM Zeiss Merlin Compact.

GC-MS assay of the wax content

The leaves or sheaths of 6-week-old rice plants were cut and photographed immediately. Then Adobe Photoshop CS6 was used to calculate the leaf area of each sample. 3 ml chloroform that preheated to 60℃ was added into a 4 ml sample bottle, the clipped leaves or leaf sheaths were immersed in chloroform for 30 s, and 10 µg n-tetracosane (C24, TCI) was added as the internal standard. The bottles were dried using a nitrogen blower, the samples were dissolved in 100 µl pyridinium, and then 30 µl silanizing agent (BSTFA-TMCS (99:1), TCI) was added. The samples were taken out and cooled to room temperature after reaction for 1 h in a 70℃ incubator, and then transferred to a new sample vial. An Agilent 7890B GC system coupled with an Agilent 5977 MS were used. GC was performed on a HP-5MS column (Agilent, 30 m × 0.25 mm i.d., 0.25 µm film thickness, 5% phenyl methyl siloxane stationary phase). The carrier gas was helium (purity > 99.999%) at a constant flow rate of 1.0 ml/min. The GC oven temperature was programmed from an initial temperature of 50°C for 2 min, ramped at 5°C/min to 300°C, and held at 300℃ for 15min. The mass range was recorded from m/z 30 to m/z 350. Electron energy was kept at 70 eV, and the source temperature and the quads temperature were kept at 230°C and 150°C, respectively. The peaks were identified based on comparison with standards and the National Institute of Standards and Technology library (NIST, version 14). The peak areas were deduced by cumulative scoring, and the contents of each ingredient were deduced through comparison with C24. The final values were shown as unit leaf area.

RNA isolation and Quantitative real–time RT–PCR (qRT–PCR) analysis

For verification of transgenic plants, seedlings were used. Total RNAs were extracted using TRIzol (Life technologies, USA) and reverse transcribed using the First Strand cDNA Synthesis Kit (Toyobo). qRT–PCR was performed with the SYBR Green Real–time PCR Master Mix Kit (Toyobo), cDNA was synthesized from 1µg of total RNA and 1µl of cDNA was used as template for real–time analysis. The rice actin (LOC_Os03g50885) and ubiquitin (LOC_Os01g22490) were used as reference genes to normalize expression levels. Data from three biological repeats were collected, and the mean value with standard error was plotted.

For gene expression assay responsive to BPH, around 5-week-old rice seedlings were individually infested with 12 second-instar BPH nymphs that had been starved for 2 h, leaf sheaths and leaves were collected after 0, 2, 4, 8, 12 and 24 h for RNA extraction, reverse transcription and qRT–PCR.

For gene expression assay responsive to drought, around 3–week–old rice seedlings that were cultivated in the rice nutrient solution (Yoshida) were directly cut off water, leaf sheaths and leaves were collected after 0, 0.5, 1, 2, 4, 6, 9 and 12 h for RNA extraction, reverse transcription and qRT–PCR.

All the primer sequences used in this study were listed in Table S2.

Luciferase Complementation Imaging assay (LCI)

The full-length cDNA of the ACL1 gene was cloned into the KpnⅠ and SalⅠ sites of pCAMBIA1300-CLuc. While the full-length cDNA of ROC1-8 coding sequences were respectively cloned into the KpnⅠ and SalⅠ sites of pCAMBIA1300-NLuc. All the constructs for test were transformed into A. tumefaciens GV3101 (pSoup-P19). Agrobacterium cells were re-suspended in infection solution (10 mM MES, 10 mM MgCl₂ and 200 µM acetosyringone) at OD600 = 1.0. The prepared suspensions were infiltrated into N. benthamiana leaves. After 2 days, the LUC signal was detected using a CCD camera and a 150 mg/mL luciferin solution.

For competitive LCI, the full-length cDNA of the ROC4 and ROC5 were cloned into the KpnⅠ and SalⅠ sites of pCAMBIA1300-CLuc and pCAMBIA1300-NLuc respectively. The full-length cDNA of the ACL1 gene was constructed into the XbaⅠ and KpnⅠ sites of p1301-35S-Nos, and p1301-35S-Nos empty vector was used as the control. The recombinant vector and the empty plasmid pGreenII 0800-LUC were transformed into Agrobacterium GV3101. The culture, injection and signal detection processes were similar. The double luciferase reporter gene analysis system (Promega, Madison, WI) was used to determinate the enzyme activity of LUC and REN. Five biological replicates were measured for each sample.

Co-IP Assay

The full-length cDNA of ACL1 was fused with FLAG tag, while the full-length cDNA of ROC1-8 were respectively fused with the GFP tag. The recombinant and control vectors were transformed into Agrobacterium GV3101. The culture and infiltration processed were similar. Injected N. benthamiana leaves were harvested after 3 d and frozen in liquid nitrogen. Soluble proteins were extracted with NB1 buffer (50 mM Tris-MES (pH8.0), 500 mM sucrose, 1 mM MgCl₂, 10 mM EDTA, 5 mM DTT, 1 mM PMSF, Cocktail). Immunoprecipitation was performed with anti-FLAG-affinity beads (Sigma-Aldrich). Lysates were incubated with the prewashed beads for 3 h. Then the beads were washed 6 times and solubilized in an appropriate volume of extraction buffer. The fusion proteins were detected by immunoblotting using monoclonal anti-FLAG M2 antibody (Sigma-Aldrich) and monoclonal antibody anti-GFP (Huabio).

For the competitive Co-IP, the full-length cDNA of ACL1 was fused with GFP, while the full-length cDNA of the ROC1-8 encoding sequence were fused with FLAG and HA respectively. The following processes were same to the Co-IP processes, except that the fusion proteins were detected by immunoblotting using monoclonal anti-FLAG M2 antibody (Sigma-Aldrich), monoclonal antibody anti-GFP (Huabio) and HA tag Polyclonal antibody (Proteintech).

Bimolecular fluorescence complementation (BIFC)

The cDNA of the coding region of ACL1 was cloned into the KpnⅠ and SalⅠ sites of pCAMBIA1300-35S-cYFP. Similarly, the cDNA sequences of the coding region of ROC1-8 were cloned into the KpnⅠ and SalⅠ sites of pCAMBIA1300-35S-nYFP. The recombinant and control vectors were transformed into Agrobacterium GV3101 respectively. The culture and infiltration processes were usual. 2 days later, the signals of YFP and mCherry were detected using confocal microscopy (Leica TSC SP8 STED 3X).

Y2H

Y2H was carried out as described (57).

Primer sequences

All the oligo sequences used in this study were listed in Table S2.

Author contributions

X.M. and Z.S. conceived and supervised the project. X.M., Z.S. and Z.T. designed the experiments. Z.T., L.Z. and B.S. performed the experiments. H.L., X.L. and D.L. discussed results and offered advices. Z.T., W.H. and S.W. analyzed the data. Z.S. wrote and revised the manuscript. X.M. revised the manuscript.

Competing interest statements

The authors declare no competing financial interests

Du B et al (2009) Identification and characterization of Bph14, a gene conferring resistance to brown planthopper in rice. Proc Natl Acad Sci USA 106(52):22163–22168
Cheng X et al (2013) A rice lectin receptor-like kinase that is involved in innate immune responses also contributes to seed germination. The Plant journal: for cell and molecular biology 76(4):687–698
Tamura Y et al (2014) Map-based cloning and characterization of a brown planthopper resistance gene BPH26 from Oryza sativa L. ssp. indica cultivar ADR52. Sci Rep 4:5872
Liu Y et al (2015) A gene cluster encoding lectin receptor kinases confers broad-spectrum and durable insect resistance in rice. Nat Biotechnol 33(3):301–305
Wang Y et al (2015) Map-based cloning and characterization of BPH29, a B3 domain-containing recessive gene conferring brown planthopper resistance in rice. J Exp Bot 66(19):6035–6045
Ji H et al (2016) Map-based Cloning and Characterization of the BPH18 Gene from Wild Rice Conferring Resistance to Brown Planthopper (BPH) Insect Pest. Sci Rep 6:34376
Ren J et al (2016) Bph32, a novel gene encoding an unknown SCR domain-containing protein, confers resistance against the brown planthopper in rice. Sci Rep 6:37645
Zhao Y et al (2016) Allelic diversity in an NLR gene BPH9 enables rice to combat planthopper variation. Proc Natl Acad Sci USA 113(45):12850–12855
Guo J et al (2018) Bph6 encodes an exocyst-localized protein and confers broad resistance to planthoppers in rice. Nat Genet 50(2):297–306
Shi S et al (2021) Bph30 confers resistance to brown planthopper by fortifying sclerenchyma in rice leaf sheaths. Mol Plant 14(10):1714–1732
Zhou C et al (2021) Balancing selection and wild gene pool contribute to resistance in global rice germplasm against planthopper. J Integr Plant Biol 63(10):1695–1711
Ling Y, Weilin Z (2016) Genetic and biochemical mechanisms of rice resistance to planthopper. Plant Cell Rep 35(8):1559–1572
Hogenhout SA, Bos JI (2011) Effector proteins that modulate plant–insect interactions. Curr Opin Plant Biol 14(4):422–428
Guo J et al (2023) A tripartite rheostat controls self-regulated host plant resistance to insects. Nature 618(7966):799–807
Hao P et al (2008) Herbivore-induced callose deposition on the sieve plates of rice: an important mechanism for host resistance. Plant Physiol 146(4):1810–1820
Raffaele S, Leger A, Roby D (2009) Very long chain fatty acid and lipid signaling in the response of plants to pathogens. Plant Signal Behav 4(2):94–99
Rahman T et al (2021) Dissecting the Roles of Cuticular Wax in Plant Resistance to Shoot Dehydration and Low-Temperature Stress in Arabidopsis. Int J Mol Sci 22(4)
Borisjuk N, Hrmova M, Lopato S (2014) Transcriptional regulation of cuticle biosynthesis. Biotechnol Adv 32(2):526–540
Seo PJ et al (2011) The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in Arabidopsis. Plant Cell 23(3):1138–1152
Zhu X, Xiong L (2013) Putative megaenzyme DWA1 plays essential roles in drought resistance by regulating stress-induced wax deposition in rice. Proc Natl Acad Sci USA 110(44):17790–17795
Wang Z et al (2018) The E3 Ligase DROUGHT HYPERSENSITIVE Negatively Regulates Cuticular Wax Biosynthesis by Promoting the Degradation of Transcription Factor ROC4 in Rice. Plant Cell 30(1):228–244
Islam MA, Du H, Ning J, Ye H, Xiong L (2009) Characterization of Glossy1-homologous genes in rice involved in leaf wax accumulation and drought resistance. Plant Mol Biol 70(4):443–456
Zhou L et al (2013) Rice OsGL1-6 is involved in leaf cuticular wax accumulation and drought resistance. PLoS ONE 8(5):e65139
Wang Y et al (2012) An ethylene response factor OsWR1 responsive to drought stress transcriptionally activates wax synthesis related genes and increases wax production in rice. Plant Mol Biol 78(3):275–288
Shepherd T, Wynne Griffiths D (2006) The effects of stress on plant cuticular waxes. New Phytol 171(3):469–499
Fang Y, Xiong L (2015) General mechanisms of drought response and their application in drought resistance improvement in plants. Cell Mol Life Sci 72(4):673–689
Todaka D, Shinozaki K, Yamaguchi-Shinozaki K (2015) Recent advances in the dissection of drought-stress regulatory networks and strategies for development of drought-tolerant transgenic rice plants. Front Plant Sci 6:84
Zhang GH, Xu Q, Zhu XD, Qian Q, Xue HW (2009) SHALLOT-LIKE1 is a KANADI transcription factor that modulates rice leaf rolling by regulating leaf abaxial cell development. Plant Cell 21(3):719–735
Sirault XR, Condon AG, Wood JT, Farquhar GD, Rebetzke GJ (2015) Rolled-upness: phenotyping leaf rolling in cereals using computer vision and functional data analysis approaches. Plant methods 11:52
Li L et al (2010) Overexpression of ACL1 (abaxially curled leaf 1) increased Bulliform cells and induced Abaxial curling of leaf blades in rice. Mol Plant 3(5):807–817
Xu Y et al (2014) Overexpression of OsZHD1, a zinc finger homeodomain class homeobox transcription factor, induces abaxially curled and drooping leaf in rice. Planta 239(4):803–816
Zou LP et al (2011) Leaf rolling controlled by the homeodomain leucine zipper class IV gene Roc5 in rice. Plant Physiol 156(3):1589–1602
Xu Y et al (2021) Heterodimer formed by ROC8 and ROC5 modulates leaf rolling in rice. Plant Biotechnol J 19(12):2662–2672
Zhao SQ, Hu J, Guo LB, Qian Q, Xue HW (2010) Rice leaf inclination2, a VIN3-like protein, regulates leaf angle through modulating cell division of the collar. Cell Res 20(8):935–947
Li C et al (2016) OsLBD3-7 Overexpression Induced Adaxially Rolled Leaves in Rice. PLoS ONE 11(6):e0156413
Shi Z et al (2007) Over-expression of rice OsAGO7 gene induces upward curling of the leaf blade that enhanced erect-leaf habit. Planta 226(1):99–108
Hu J et al (2010) Identification and characterization of NARROW AND ROLLED LEAF 1, a novel gene regulating leaf morphology and plant architecture in rice. Plant Mol Biol 73(3):283–292
Fang L et al (2012) Rolling-leaf14 is a 2OG-Fe (II) oxygenase family protein that modulates rice leaf rolling by affecting secondary cell wall formation in leaves. Plant Biotechnol J 10(5):524–532
Li YY et al (2016) Overexpression of OsHox32 Results in Pleiotropic Effects on Plant Type Architecture and Leaf Development in Rice. Rice 9(1):46
Nalam V, Louis J, Shah J (2019) Plant defense against aphids, the pest extraordinaire. Plant science: an international journal of experimental plant biology 279:96–107
May DG, Scott KL, Campos AR, Roux KJ (2020) Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation. Cells 9(5)
Zhang K, Li Y, Huang T, Li Z (2022) Potential application of TurboID-based proximity labeling in studying the protein interaction network in plant response to abiotic stress. Front Plant Sci 13:974598
Chew W, Hrmova M, Lopato S (2013) Role of Homeodomain leucine zipper (HD-Zip) IV transcription factors in plant development and plant protection from deleterious environmental factors. Int J Mol Sci 14(4):8122–8147
Sun J et al (2020) HD-ZIP IV gene Roc8 regulates the size of bulliform cells and lignin content in rice. Plant Biotechnol J 18(12):2559–2572
Matschi S et al (2020) Structure-function analysis of the maize bulliform cell cuticle and its potential role in dehydration and leaf rolling. Plant direct 4(10):e00282
Seo PJ, Park CM (2011) Cuticular wax biosynthesis as a way of inducing drought resistance. Plant Signal Behav 6(7):1043–1045
Bi H et al (2017) Wheat drought-responsive WXPL transcription factors regulate cuticle biosynthesis genes. Plant Mol Biol 94(1–2):15–32
Wu R, Citovsky V (2017) Adaptor proteins GIR1 and GIR2. I. Interaction with the repressor GLABRA2 and regulation of root hair development. Biochem Biophys Res Commun 488(3):547–553
Walker AR et al (1999) The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell 11(7):1337–1350
Khosla A et al (2014) HD-Zip Proteins GL2 and HDG11 Have Redundant Functions in Arabidopsis Trichomes, and GL2 Activates a Positive Feedback Loop via MYB23. Plant Cell 26(5):2184–2200
Yang C et al (2011) A regulatory gene induces trichome formation and embryo lethality in tomato. Proc Natl Acad Sci USA 108(29):11836–11841
Wang Z et al (2022) NtCycB2 negatively regulates tobacco glandular trichome formation, exudate accumulation, and aphid resistance. Plant Mol Biol 108(1–2):65–76
Schrick K, Ahmad B, Nguyen HV (2023) HD-Zip IV transcription factors: Drivers of epidermal cell fate integrate metabolic signals. Curr Opin Plant Biol 75:102417
Bonaventure G (2012) Perception of insect feeding by plants. Plant Biol 14(6):872–880
Ji C et al (2022) The O2-ZmGRAS11 transcriptional regulatory network orchestrates the coordination of endosperm cell expansion and grain filling in maize. Mol Plant 15(3):468–487
Dai Z et al (2018) Modulation of plant architecture by the miR156f-OsSPL7-OsGH3.8 pathway in rice. J Exp Bot 69(21):5117–5130
Sun B et al (2023) A novel transcriptional repressor complex MYB22-TOPLESS-HDAC1 promotes rice resistance to brown planthopper by repressing F3'H expression. New Phytol 239(2):720–738

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ACL1-HD-Zip IV complex revealed common mechanism in rice response to brown planthopper and drought

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Introduction

Results

Similar cytological response of rice plants to drought and BPH infestation

ACL1 interacted with almost all ROC proteins in rice

ACL1 compete with ROC4-ROC4 and ROC5-ROC5 homodimers, and ROC4/5 heterodimer

ACL1-ROCs complexes synergistically regulated drought and BPH resistance

Discussion

Material and Methods

Plant species and growth conditions

Plasmid construction and plant transformation

Calculation of water loss efficiency

Chlorophyll leaching assay

TurboID-mediated proximity labelling proteomics

Sequence Database Searching and Data Analysis

Phylogenetic analysis

Western blot

BPH resistance detection and measurements

Anatomical analysis

Histochemical GUS staining

Drought tolerance detection methods

Scanning electron microscope (SEM) assay of the leaf/sheath surface

GC-MS assay of the wax content

Luciferase Complementation Imaging assay (LCI)

Co-IP Assay

Bimolecular fluorescence complementation (BIFC)

Y2H

Primer sequences

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1