New Results

Disruption of the rice 4-DEOXYOROBANCHOL HYDROXYLASE unravels specific functions of canonical strigolactones

Guan-Ting Erica Chen, Jian You Wang, Cristina Votta, Justine Braguy, Muhammad Jamil, Gwendolyn K Kirschner, Valentina Fiorilli, Lamis Berqdar, Aparna Balakrishna, Ikram Blilou, Luisa Lanfranco, Salim Al-Babili
doi: https://doi.org/10.1101/2023.04.03.535333

Abstract

Strigolactones (SLs) regulate many developmental processes, including shoot-branching/tillering, and mediate rhizospheric interactions. SLs are structurally diverse, divided into a canonical and a non-canonical sub-family. To better understand the biological function of particular SLs, we generated CRISPR/Cas9 mutants disrupted in OsMAX1-1400 or OsMAX1-1900, which encode cytochrome P450 enzymes (CYP711A clade) contributing to SL diversity. The disruption of OsMAX1-1900 did neither affect the SL pattern nor plant architecture, indicating a functional redundancy. In contrast, disruption of OsMAX1-1400 activity, a 4-deoxyorobanchol hydroxylase, led to a complete lack of orobanchol and an accumulation of its precursor 4-deoxyorobanchol (4DO), both of which are a canonical SLs common in different plant species, accompanied by higher levels of the non-canonical methyl 4-oxo-carlactonoate (4-oxo-MeCLA). Os1400 mutants showed also shorter plant height, panicle and panicle base length, but did not exhibit a tillering phenotype. Hormone quantification and transcriptome analysis revealed elevated auxin levels and changes in the expression of auxin-related, as well as of SL biosynthetic genes. Interestingly, the Os900/1400 double mutant lacking both orobanchol and 4DO did not show the observed Os1400 architectural phenotypes, indicating that they are a result of 4DO accumulation. A comparison of the mycorrhization and Striga seed germinating activity of Os900, Os900/1400, and Os1400 loss-of-function mutants demonstrates that the germination activity positively correlates with 4DO content while disrupting OsMAX1-1400 negatively impact mycorrhizal symbiosis. Taken together, our paper deciphers the biological function of canonical SLs in rice and depicts their particular contributions to establishing architecture and rhizospheric communications.

Introduction

The apocarotenoid-derived strigolactones (SLs) are a novel class of plant hormones induced under low phosphate (Pi) condition that inhibits shoot branching/tillering (Gomez-Roldan et al., 2008; Umehara et al., 2008) and regulates other plant processes and features, including root development, stem thickness, and leaf senescence (Al-Babili & Bouwmeester, 2015; Fiorilli et al., 2019). Before being recognized as a plant hormone, SLs were first discovered to be the germinating stimulants for root parasitic weeds, such as Orobanche and Striga spp. (Cook et al., 1966), and later found as an initiation signal for establishing beneficial arbuscular mycorrhizal fungi (AMF) symbiosis, by inducing the AMF hyphal branching (Akiyama et al., 2005; Lanfranco et al., 2018). In addition, SLs can orchestrate plant architecture as SL-deficient plants, such as rice d17, consistently exhibit a distinct phenotype comprising higher numbers of branches/tillers, shorter shoot and primary root length when compared to wild-type (WT) (Al-Babili & Bouwmeester, 2015; Gomez-Roldan et al., 2008; Morris et al., 2001), suggesting that SLs are more than rhizospheric signals (Wang et al., 2022a).

SLs are characterized based on their exclusive chemical structure, the lactone ring (D-ring; Fig.1A). The latter is linked to an enol-ether bridge, essential for the SL biological activity (Yoneyama et al., 2018). According to the presence or absence of the BC-ring (Fig.1A), they are classified into canonical and non-canonical SLs, respectively (Al-Babili & Bouwmeester, 2015; Wang et al., 2021). In general, the evolutionarily-conserved SL biosynthetic pathway in land plants starts specifically with 9-cis-β- carotene after its isomerization by DWARF27 (D27) (Abuauf et al., 2018). Then, two carotenoid cleavage dioxygenases (CCDs), CCD7 (D17) and CCD8 (D10), cleave successively 9-cis-β-carotene into carlactone (CL), the core intermediate of SL biosynthesis in planta (Alder et al., 2012; Bruno et al., 2014; Chen et al., 2022; Seto et al., 2014; Wang et al., 2021).

Figure 1.
Figure 1. Generation of the Os1400-KO lines by CRISPR-Cas9 system.

(A) Scheme of the biosynthesis of the rice canonical SLs (the detailed SL biosynthesis pathway depicted in fig. S1. (B) The structure of the Os1400 gene and the sequences of the two CRISPR-Cas9 target sites indicated by red arrows 3 and 4. Details of the CRISPR-mediated mutations of the two KO lines, Os1400-12SIII and Os1400-12SIV, are reported in FigS2. (C) Analysis of SLs in root exudates and root tissues of WT, Os900-KO line, Os900/1400-KO line, Os1400-KO lines, and d17 mutant grown under constant low-Pi conditions. The data are presented as means ± SD for the number of biological replicates n=4 for (C). Significant values determined by one-way ANOVA are shown with different letter (P < 0.05) when compared to WT, and asterisks indicate statistically significant differences as compared to control by two tailed paired Student’s t-test (*p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001). Abbreviation: 4DO, 4-Deoxyorobanchol

Until now, more than 35 natural SLs, with different chemical structures, are identified in Plantae (Yoneyama et al., 2018). Their structural diversity arose from the CL catabolism by MORE AXILLARY GROWTH1 (MAX1) from the cytochrome P450 monooxygenase (CYP) 711A family (Booker et al., 2005; Cardoso et al., 2014; Lazar & Goodman, 2006), and the recently identified CYP722C, CYP712G1, CYP706C37 clades (Li et al., 2023; Wakabayashi et al., 2019; Wang et al., 2022c). In rice, OsMAX1-900 repeatedly oxygenates CL to produce the canonical SL 4-deoxyorobanchol (4DO) in vivo (Ito et al., 2022). Based on in vitro studies and expression in Nicotiana benthamiana, 4DO is further hydroxylated into orobanchol (Oro) by another CYP711A enzyme, OsMAX1-1400, (Zhang et al., 2014).

The biological relevance of such a big family of compounds is yet to be discovered by the scientific community. Therefore, we started our investigation in rice and created CRISPR-mediated mutant lines of OsMAX1s. Our previous study surprisingly showed that mutants created by targeting OSMAX1-900 do not display a typical SL-deficiency phenotype (Ito et al., 2022). This revealed that canonical SLs are not the major tillering regulators, and hence the biological functions of canonical SLs inside plants remain elusive. To have a comprehensive view, in this work we aimed to understand the biological roles of canonical SLs in rice by studying mutants defective of Osmax1-1400, the other rice enzyme involved in canonical SLs synthesis.

Results and Discussion

Characterization of rice MAX1 homologs

To investigate the biological functions of canonical SLs in rice, we generated several mutant lines using CRISPR/Cas9 technology. We targeted the biosynthesis of canonical SLs by generating biallelic homozygote Osmax1-1400 (Os1400-12SIII and -12SIV, Fig. S2). We also generated Osmax1-1900 (Os1900-13DI and -13EI, Fig. S3) rice mutant lines to understand whether the MAX1-1900 would contribute to SL biosynthesis in planta, as MAX1-1900 is phylogenetically from a distinct clade, and only shown to weakly convert CL into CLA in vitro (Marzec et al., 2020; Yoneyama et al., 2018).

First, we quantified the known SLs in these mutants’ roots and root exudates, together with Os900 and SL-deficient d17, using Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS) under phosphate (Pi) starvation conditions (Fig. 1C; Fig. 1D; Fig. S4). As expected, no SLs were detected in d17, while the CRISPR/Cas9-induced deletion impaired the biosynthesis of the canonical SLs, which was evidenced by the absent Oro in the roots and root exudates of Os1400 mutants (Fig. 1C). This confirmed OsMAX1-1400 as the 4DO hydroxylase in planta (Zhang et al., 2014). Interestingly, the accumulation of two SLs, 4DO and the putative methyl 4-oxo-carlactonoate (4-oxo-MeCLA), produced through OsMAX1-900 (Ito et al., 2022), were doubled in the root exudates of Os1400 when compared to wild-type (WT) plants (Fig. 1D). The production of 4-oxo-MeCLA is likely through an additional step by an uncharacterized methyltransferase (Ito et al., 2022), suggesting that 4DO metabolism is exclusive by OsMAX1-1400. Moreover, the amount of putative non-canonical SLs, CL+30 and oxo-CL (Ito et al., 2022; Wang et al., 2022a), in the root exudates of Os1400 were comparable to that of WT (Fig. S4), affirming the mutation of Os1400 mainly affects the canonical SL Oro metabolism.

In contrast, we did not observe remarkable changes in SL composition in the root tissues and root exudates between Os1900 mutants and WT (Fig. S5), indicating that the role of OsMAX1-1900 might be functionally redundant in SL biosynthesis in plants. This was supported by no consistently significant differences in the shoot and root phenotypes grown under normal or low Pi conditions as well as Striga germination activity, when compared to the WT (Fig. S6, Fig. S7).

Accumulation of 4-deoxyorobanchol negatively regulates rice plant growth and development

Recently, we reported that rice lacking canonical SLs, 4DO and Oro, displays shoot phenotypes comparable to WT, revealing that canonical SLs are not the major players in regulating shoot architectures (Ito et al., 2022). However, rice has duplicated MAX1 genes during evolution (Marzec et al., 2020); intriguingly, OsMAX1-1400 is the only rice MAX1 acting in the final step of canonical SL biosynthesis (Fig. 1A) (Al-Babili & Bouwmeester, 2015; Zhang et al., 2014). Therefore, we suspected that Os1400 and its downstream metabolite Oro might hold biological importance in plants. To test this hypothesis, we phenotyped Os1400, Os900, d17, and WT rice under soil and hydroponic conditions. Expectedly, the MAX1 mutants, grown in greenhouse, did not showed high tiller numbers with dwarf appearance, the typical SL-deficit (d17) phenotype (Fig. 2A). Instead, the plant height, panicle length, and length of the panicle base (the distance from flag leaf auricle to the panicle base on the primary branch) of Os1400 mutants were significantly shorter than that of WT and Os900 (Fig. 2B). No differences were observed in the total number of tillers, productive tillers, and average panicle numbers under greenhouse conditions (Fig. S8). The shorter shoot phenotype was also detected in the hydroponically grown Os1400 mutants under both normal and low-Pi conditions (Fig. S9; Fig. S10).

Figure 2.
Figure 2. Phenotypic characterization of OsMAX1 mutants.

(A and B) Shoot phenotypes of WT, Os900-KO line, Os900/1400-KO line, Os1400-KO lines, and d17 mutant grown in soil. Scale bar, 10cm. (C and D) Panicle phenotypes of WT, Os900-KO line, Os900/1400-KO line, Os1400-KO lines, and d17 mutant. Scale bar, 5 cm The data are all presented as means ± SD for the number of biological replicates n=6. Significant values determined by one-way ANOVA are shown with different letter (P < 0.05) when compared to WT, and asterisks indicate statistically significant differences as compared to control by two tailed paired Student’s t-test (*p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001).

It seems that the accumulation of 4DO could be an endogenous unfavorable/toxic signal to rice plants. To confirm the assumption that the shorter shoot phenotype is caused by 4DO accumulation, we exogenously supplied 4DO at 300 nM and 900 nM under normal growth conditions -without triggering SL biosynthesis- to WT and Os1400 mutants. Interestingly, 300 nM 4DO treatment remarkably increased the difference of shoot length in Os1400 mutants compared to untreated WT; however, with 900 nM 4DO application, the shoot length in the WT was also suppressed, suggesting that 4DO is a negative regulator of shoot growth and development in rice (Fig. S11). On the other hand, we observed a longer root length tendency in Os1400 than WT (Fig. S10) under low-Pi conditions, while a decreased tendency in root length and root diameter under normal conditions (Fig. S9; Fig. S12). As SL biosynthesis is triggered by Pi-starvation, we postulated that Pi content might influence the amount of 4DO accumulated in the plant, leading to the different tendency in root length under various growth conditions. Indeed, exogenous 4DO application under normal conditions, mimicking SL biosynthesis, enhanced the root length in all treated rice plants grown in hydroponics (Fig. S11).

Next, to check our rescue hypothesis by reducing the level of 4DO accumulated in Os1400 mutants, we treated Os1400 grown under normal conditions with 5 µM TIS108, a MAX1-900 and MAX1-1400 inhibitor (Ito et al., 2022). The root length and crown root numbers of mutants treated with TIS108 were restored to that of the WT (Fig. S13), providing a further evidence that the enzymatic activity on 4DO by OsMAX1-1400 is important for an optimal rice root development. In fact, the root-released 4DO level of Os1400 mutants was suppressed upon TIS108 treatment, accompanied by the accumulation of the putative non-canonical SLs, CL+30 and oxo-CL (Fig. S14) (Ito et al., 2022; Wang et al., 2022a). Although the shoot length of Os1400 mutants no longer showed difference to the WT when treated with TIS108, we observed that two-week application of TIS108 unexpectedly decreased the shoot length of WT (Fig. S13), probably due to unspecific compound effects on other CYPs. Hence, to specifically understand the biological roles of canonical SL biosynthesis, we generated a homozygous Osmax1-900/1400 (Os900/1400-4BI) rice double mutant line (Fig. S15), which has full disruption of canonical SLs, to compare with the TIS108 observation. Notably, the observed phenotypes of Os1400 mutants were no longer present in the double mutant in all growth conditions (Fig. 2; Fig. S8; Fig. S9; Fig. S10: Fig. S12), together with the absence of canonical SLs and the accumulation of non-canonical SLs in the roots and root exudates of Os900/1400 (Fig. 1C; Fig. 1D; Fig. S4). Sharing the same rescue mechanism as TIS treatment, suppressing both 4DO and Oro biosynthesis, Os900/1400 provides a stronger genetic evidence without the possibilities of chemical effects. These suggest that the metabolism of 4DO by OsMAX1-1400 is required for normal rice growth and development.

One previous study had contradictory results, showing a high-tillering phenotype with low canonical SLs in the Bala rice that has both a deletion of MAX1-900 and MAX1-1400 (Cardoso et al., 2014). However, our observations hold stronger genetic evidence since these genetic materials were compared within the same cultivar, while the tillering phenotype of Bala rice might be a consequence of other genetic regulations. Notably, 4-oxo-MeCLA was present in the root exudate of Os900/1400, as well as in Bala rice root exudate (Cardoso et al., 2014), indicating that the biosynthesis of this non-canonical SL is independent of MAX1-900 and MAX1-1400. Overall, we can conclude that 4DO and Oro are not tillering-inhibitory regulators, but their unbalanced metabolism negatively affects the physiological development of rice plants.

Furthermore, we investigated the rice transcriptome of the Os1400 grown under normal and low-Pi conditions compared to WT by RNAseq (Data S1). In the differentially expressed genes (DEGs), there were 1712 upregulated genes and 1465 downregulated genes under normal conditions, while 5890 upregulated genes and 3569 downregulated genes were observed under low-Pi conditions (Fig. S16). None of the DEGs related to tillering or SL biosynthesis was observed under normal conditions (Tables S1); in contrast, the transcripts of SL biosynthesis and signaling were generally downregulated under low-Pi conditions (Fig. 3A, Tables S2), suggesting that the 4DO accumulation might lead to a negative feedback loop in Os1400 mutants. Interestingly, under both conditions, we observed many downregulated auxin-related genes in Os1400 mutants (Fig. 3B, Tables S1-S2).

Figure 3.
Figure 3. Transcriptome and hormone analysis of Os1400-KO lines.

(A) Differentially expressed genes (DEGs) of SL biosynthesis and signaling related gene. Expression pattern was shown in log2FoldChange (Log2FC). Statistically significant differences indicated by adjusted p-value (*< 0.05). (B) Heat map analysis of DEGs involved Auxin pathways. AUXIN1/LIKE-AUX1 (AUX1/LAX) are major auxin influx carriers; AUXIN/indole-3-acetic acid (AUX/IAA) are transcriptional repressors; the PIN-FORMED (PIN) proteins are secondary transporters in the efflux of auxin; GH3 gene family encodes auxin-amido synthetases. Expression pattern was shown in log2FoldChange (Log2FC). Statistically significant differences indicated by adjusted p-value (*< 0.05). (C) Analysis of IAA (auxin) in root and shoot of WT, and Os1400-KO lines grown under constant low-Pi and +Pi conditions. Abbreviation: IAA, indole-3-acetic acid. The data are all presented as means ± SD for the number of biological replicates n=3 for (A and B), and n=4 for (C). Significant values determined by one-way ANOVA are shown with different letter (P < 0.05) when compared to WT, and asterisks indicate statistically significant differences as compared to control by two tailed paired Student’s t-test (*p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001).

Auxin regulates meristem activities and interplays with SLs on the root and shoot development (Su et al., 2011; Xiao et al., 2019; Yang et al., 2019); thus, we hypothesized the phenotypes observed in Os1400 mutants could be linked to auxin. We then determined the hormone content of auxin (IAA), gibberellin (GA), abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) in roots and shoot bases (root-shoot junction) of Os1400 mutants under normal and low Pi conditions. We did not detect any consistent significant difference in the levels of GAs, ABA, SA, or JA (Fig. S17), but observed a remarkable increase of IAA level in root and shoot bases in both growth conditions, compared to the WT (Fig. 3C; Fig. S17). Consistently, excess of IAA content, by either overexpression of OsPIN2 or exogenous IAA application, causes shorter shoot phenotype in rice (Liu et al., 2019; Sun et al., 2019). Furthermore, we performed 5-Ethynyl-2’-deoxyuridine (EdU) staining to visualize proliferating cells and measured the root meristem length. Yet, no clear differences were observed between Os1400 mutants and WT (Fig. S18). Accordingly, we demonstrated that the shoot phenotypes of Os1400 plants are likely due to auxin homeostasis modulation, but the role of auxin in Os1400 roots needs further investigation.

The metabolism of rice canonical SL, 4-deoxyorobanchol, is associated to rhizospheric signals

Although all SLs seem to be communicating signals in the rhizosphere (Ito et al., 2022; Wang et al., 2022a), their bioactivities - to induce AM hyphal branching and trigger parasitic seed germination - depend largely on their chemical structures (Gobena et al., 2017; Mori et al., 2016). The distinguishable root-released SL compositions in Os900, Os900/1400, and Os1400 (Fig. 1; Fig. S1) make them good candidates to investigate the possible functions of these metabolites in the rhizosphere. We then tested the germination activity of Os900, Os1400, and Os900/1400 root exudates on Striga hermonthica seeds. Compared to WT exudates, we observed more than 40% decrease in the Striga germination of Os900 exudates and an even lower germination rate of Os900/1400 exudates (Fig. 4A), indicating that 4-oxo-MeCLA is not a predominate germinating signal for Striga (Fig. S4). Although the stimulation activity on Striga germination of Os1400 exudates was comparable to the WT at 1:1 dilution, we found an increased tendency at 1:3 dilution (Fig. 4A; Fig. S19A). This reveals that 4DO is a stronger germination cue for Striga seeds than Oro, supported experimentally by 10 µM Oro exerting less seed germination activity than 1 µM rac-GR24, a 4DO-like SL analog (Fig. S19B).

Figure 4.
Figure 4. Assessment of rhizospheric interactions.

Effect of Os1400-KO lines on (A) the germination of root parasitic weed Striga and (B and C) the arbuscule formation.. The R. irregularis colonization was quantified by measuring the expression of an AM marker gene (OsPT11) (B). Arbuscule formation at 10 dpi and 40 dpi. Arrows indicate arbuscule containing cells. Scale bars, 50 µm (C). The data are all presented as means ± SD for the number of biological replicates n=4 for (A), and n≥3 for (B). Significant values determined by one-way ANOVA are shown with different letter (P < 0.05) when compared to WT, and asterisks indicate statistically significant differences as compared to control by two tailed paired Student’s t-test (*p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001).

Moreover, Oro seems to be the preferable signal for AM symbiosis (Mori et al., 2016), we then investigated the role of 4DO and Oro in establishing AM symbiosis by comparing Os900, Os1400, Os900/1400, and WT. For this purpose, we examined the root colonization with the AMF Rhizophagus irregularis after 10- and 40-days post inoculation (dpi) and measured the transcript level of OsPT11, a specific AM inducible Pi-transporter gene (Güimil et al., 2005). At 10 dpi, there was a delay in colonization of all mutant roots compared to WT roots; whereas, at 40 dpi, the colonization of Os1400 and Os900/1400 mutants was surprisingly much lower than that of the WT and Os900 (Fig. 4B; Fig. S20), which indicates that MAX1-1400 is crucial for maintaining AM colonization. Additionally, absence of MAX1-1400 did not influence the intraradical fungal structures of the arbuscules. Instead, they appeared well developed and regularly branched (Fig. 4C), suggesting that MAX1-1400 does not affect the fungal morphology. Therefore, we can speculate that MAX1 duplication is highly associated with AM symbiosis; besides, the decreased colonization level of Os900/1400 mutant further supported that the reduced AM symbiotic pattern upon TIS108 application in WT plants (Ito et al., 2022) might be a consequence of MAX1-1400 suppression.

Taken together, the generation of the CRISPR-mediated Os1400 rice mutants allowed us to reveal the biological roles of canonical SLs in plant growth and development, and as well confirmed OsMAX1-1400 as the 4DO hydroxylase in planta. Finally, we can conclude that the canonical SLs, 4DO and Oro, are critical rhizospheric signals for the interaction with AMF and root parasitic plants, and the MAX1 duplication might be also evolutionary required for beneficial symbiosis in rice. Importantly, knocking out entire canonical SLs, without damaging plant architecture in the Os900/1400 double mutant, seems promising to reduce the yield loss caused by Striga and other root parasitic plants; thus, offering an alternative way to improve global food security.

Material and Methods

Plant material and growth conditions

Oryza sativa Nipponbare d17 (Butt et al., 2018), max1-900 (Ito et al., 2022), max1-1400, max1-900/1400, max1-1900, and WT rice plants were grown under controlled conditions (a 12 h photoperiod, 200-µmol photons m−2 s−1 and day/night temperature of 27/25 °C). All rice seeds were first surface-sterilized in a 50% sodium hypochlorite solution with 0.01 % Tween-20 for 15 min, then rinsed with sterile water, before being germinated in the dark overnight. The pre-germinated seeds were placed on Petri dishes containing half-strength liquid Murashige and Skoog (MS) medium and incubated in a growth chamber for 7 days. Thereafter, the seedlings were transferred into 50mL black falcon tubes filled with half-strength modified Hoagland nutrient solution with adjusted pH to 5.8. The nutrient solution consisted of 5.6 mM NH4NO3, 0.8 mM MgSO4·7H2O, 0.8 mM K2SO4, 0.18 mM FeSO4·7H2O, 0.18 mM Na2EDTA·2H2O, 1.6 mM CaCl2·2H2O, 0.8 mM KNO3, 0.023 mM H3BO3, 0.0045 mM MnCl2·4H2O, 0.0003 mM CuSO4·5H2O, 0.0015 mM ZnCl2, 0.0001 mM Na2MoO4·2H2O and 0.4 mM K2HPO4·2H2O.

Generation of Os1900, Os1400, and Os1400/900 plants

Two guide RNAs [gRNAs; single gRNA3 (sgRNA3), 5’ -tgcgaacaggttgaaattgg-3’ and sgRNA4, 5’ -ctcgagtttcagtactcgat-3’] were designed to target the rice (O. sativa L. ssp. japonica cv. Nipponbare) OsMAX1-1400 (Os01g0701400 /AP014957) gene. By using Golden Gate cloning, the tRNA-gRNA-Cas9 cassette was assembly into the pRGEB32 binary vector that has hygromycin resistance gene for selection. With mature seeds, Nipponbare calli were induced and transformed with Agrobacterium tumefaciens EHA105 culture containing the plasmid of interest. Later, shoots and roots were regenerated in a Percival growth chamber (CLF Plant Climatics GmbH, model CU 36L5), and then transferred to soil and grown in a greenhouse at 28°C day/22°C night.

Genomic DNA was extracted from the rice young leaves, and plant transgenicity and mutagenicity were demonstrated. Through polymerase chain reaction (PCR) amplification, the transgenic plants were recognized when the pRGEB32-specific primers, pRGEB32-F (5′-ccacgtgatgtgaagaagtaagataaactg-3′) and pRGEB32-R (5′-gataggtttaagggtgatccaaattgagac-3′), bind to the surrounding region of the insertion sites in the pRGEB32 vector. For identifying CRISPR-mediated mutations, the DNA region that includes the sgRNA target sites were amplified using genome specific primers Os1400 sg3-sg4 F (5’-tcagcgcgctcacttacga -3’) and Os1400 sg4 F1 (5’-atcccaagaacttcccggag-3’).

Hydroponic culture of rice seedlings

The hydroponic culture system is built with 50-mL black falcon tubes with punctured caps inserted with a 1.5-ml bottomless Eppendorf tube in the center. Nutrition solution, containing normal (+Pi) or low 0.004 mM K2HPO3·3H2O (lowPi), was applied to the transferred 1-week old seedlings for the following 2 weeks. The solutions were changed every 3 days, and adjusted to pH 5.8 every time before applying. All plants were kept in the solution for 3 weeks, except the plants for 4DO application and EdU staining were 10-days seedlings.

Phenotyping in pots under greenhouse conditions

To study the phenotype of Os1400 and Os900/1400 mutants, seedlings were transferred into pots packed with soil. The soil were soaked with half-strength modified Hoagland nutrient solution in advance. The nutrient solution comprised 5.6 mM NH4NO3, 0.8 mM MgSO4.7H2O, 0.8 mM K2SO4, 0.18 mM FeSO4.7H2O, 0.18 mM Na2EDTA.2H2O, 1.6 mM CaCl2.2H2O, 0.8 mM KNO3, 0.023 mM H3BO3, 0.0045 mM MnCl2.4H2O, 0.0003 mM CuSO4.5H2O, 0.0015 mM ZnCl2, 0.0001 mM Na2MoO4.2H2O, and 0.4 mM K2HPO4.2H2O. The pH of the solution was adjusted to 5.8, and the solution was applied every third day. On day 120, phenotypic data were recorded. The plants were grown in a greenhouse from February to May 2022, in Thuwal (Saudi Arabia).

Exogenous applications of 4DO and TIS108

For investigating the effect of 4DO (Olchemim, Czech Republic) on different genotypes, 1-week-old seedlings were grown hydroponically in half-strength Hoagland nutrient solution containing 0.4 mM K2HPO4·2H2O (+Pi), 300 nM or 900 nM 4DO (dissolved in acetone), or the corresponding volume of the solvent (mock; acetone) for 10 or 14 days. The solution was changed three times per week, adding the chemical at each renewal.

For investigating the effect of TIS108, 2-week-old rice seedlings were grown hydroponically in half-strength Hoagland nutrient solution containing 0.4 mM K2HPO4·2H2O (+Pi), 5 µM TIS108 (dissolved in acetone), or the corresponding volume of the solvent (mock; acetone) for 14 days. The solution was changed twice per week, adding the chemical at each renewal.

SL quantification in root tissues and exudates

Analysis of SLs in rice root exudates was performed according to the published protocol (Wang et al., 2022b). Briefly, root exudates spiked with 2 ng of GR24 were brought on a C18-Fast Reversed-SPE column (500 mg/3 mL), preconditioned with 3 mL of methanol and followed with 3 mL of water. After washing with 3 mL of water, SLs were eluted with 5 mL of acetone. Thereafter, SLs-containing fraction was concentrated to SL aqueous solution (∼500 μL), followed by 1 mL of ethyl acetate extraction. 750 μL of SL enriched fraction was dried under vacuum. The final extract was re-dissolved in 100 μL of acetonitrile: water (25:75, v:v) and filtered through a 0.22 μm filter for LC-MS/MS analysis.

SL extraction from root tissues was followed the procedure (Wang et al., 2019). Around 25 mg of lyophilized and grinded rice root tissues, spiked with 2 ng of GR24, were extracted twice with 2 mL of ethyl acetate in an ultrasound bath (Branson 3510 ultrasonic bath) for 15 min, followed by centrifugation for 8 min at 3800 rpm at 4 °C. The two supernatants were combined and dried under vacuum. The residue was dissolved in 50 μL of ethyl acetate and 2 mL of normal hexane, purifying with a Silica Cartridges SPE column (500 mg/3 mL). After washing with 3 mL of hexane, SLs were eluted in 3 mL of ethyl acetate and evaporated to dryness under vacuum. The final extract was re-dissolved in 150 μL of acetonitrile: water (25:75, v:v) and filtered through a 0.22 μm filter for LC-MS/MS analysis.

SLs were quantified by LC-MS/MS using UHPLC-Triple-Stage Quadrupole Mass Spectrometer (Thermo Scientific™ Altis™). Chromatographic separation was achieved on the Hypersil GOLD C18 Selectivity HPLC Columns (150 × 4.6 mm; 3 μm; Thermo Scientific™) with mobile phases consisting of water (A) and acetonitrile (B), both containing 0.1% formic acid, and the following linear gradient (flow rate, 0.5 mL/min): 0– 15 min, 25%–100 % B, followed by washing with 100 % B and equilibration with 25 % B for 3 min. The injection volume was 10 μL, and the column temperature was maintained at 35 °C for each run. The MS parameters of Thermo ScientificTM Altis™ were as follows: positive ion mode, ion source of H-ESI, ion spray voltage of 5000 V, sheath gas of 40 arbitrary units, aux gas of 15 arbitrary units, sweep gas of 20 arbitrary units, ion transfer tube gas temperature of 350 °C, vaporizer temperature of 350 °C, collision energy of 17 eV, CID gas of 2 mTorr, and full width at half maximum (FWHM) 0.2 Da of Q1/Q3 mass. The characteristic Multiple Reaction Monitoring (MRM) transitions (precursor ion → product ion) were 331.15→216.0, 331.15→234.1, 331.15→97.02 for 4-deoxyorobanchol; 347.14→329.14, 347.14→233.12, 347.14→ 205.12, 347.14→97.02 for orobanchol; 361.16→ 247.12, 361.16→177.05, 361.16→208.07, 361.16→97.02 for putative 4-oxo-MeCLA; 333.17→219.2, 333.17→173.2, 333.17→201.2, 333.17→97.02 for putative 4-oxo-hydroxyl-CL (CL+30); 317.17→164.08, 317.17→97.02 for putative oxo-CL (CL+14); 299.09→158.06, 299.09→157.06, 299.09→97.02 for GR24.

Quantification of plant hormones

Quantification of endogenous hormones was followed the procedure (Wang et al., 2021). Briefly, 15 mg freeze-dried ground root or shoot base tissues were spiked with internal standards D6-ABA (10 ng), D2-GA1 (10 ng), D2-IAA (10 ng), D4-SA (10 ng), and D2-JA (10 ng) along with 750 µL of methanol. The mixture was sonicated for 15 min in an ultrasonic bath (Branson 3510 ultrasonic bath), followed by centrifugation for 5 min at 14,000 × g at 4 °C. The supernatant was collected, and the pellet was re-extracted with 750 µL of the same solvent. Then, the two supernatants were combined and dried under a vacuum. The sample was re-dissolved in 100 μL of acetonitrile:water (25:75, v-v) and filtered through a 0.22 μm filter for LC–MS analysis.

Plant hormones were analyzed using LC-MS/MS using UHPLC-Triple-Stage Quadrupole Mass Spectrometer (Thermo Scientific™ Altis™). Chromatographic separation was achieved on the Hypersil GOLD C18 Selectivity HPLC Columns (150 × 4.6 mm; 3 μm; Thermo Scientific™) with mobile phases consisting of water (A) and acetonitrile (B), both containing 0.1% formic acid, and the following linear gradient (flow rate, 0.5 mL/min): 0–10 min, 15%–100 % B, followed by washing with 100 % B for 5 min and equilibration with 15 % B for 2 min. The injection volume was 10 μL, and the column temperature was maintained at 35 °C for each run. The MS parameters of Thermo ScientificTM Altis™ were as follows: positive ion mode for IAA and negative mode for GA, ABA, SA, and JA, ion source of H-ESI, ion spray voltage of 3000 V, sheath gas of 40 arbitrary units, aux gas of 15 arbitrary units, sweep gas of 0 arbitrary units, ion transfer tube gas temperature of 350 °C, vaporizer temperature of 350 °C, collision energy of 20 eV, CID gas of 2 mTorr, and full width at half maximum (FWHM) 0.4 Da of Q1/Q3 mass. The characteristic Multiple Reaction Monitoring (MRM) transitions (precursor ion → product ion) were The characteristic MRM transitions (precursor ion → product ion) were 176.2 → 130 for IAA; 263.2 → 153.1, 263.3 → 204.1, 263.3 → 219.1 for ABA; 347.2 → 259.1, 347.2 → 273 for GA1; 345.1 → 143, 345.1 → 239 for GA3; 137.1 → 93.15, 137.1 → 65.1 for SA; 209.15 → 59.05, 209.15 → 93.04 for JA; 178.2 → 132 for D2-IAA; 269.2 → 159.1 for D6-ABA; 349.1 → 261.1 for D2-GA1; 141.0 → 97.0 for D4-SA ; 211.0 → 61.0 for D2-JA.

Striga hermonthica seed germination bioassays

Striga seed germination bioassay was carried out based on the protocol (Jamil et al., 2012; Wang et al., 2022). Briefly, 10-day-old pre-conditioning Striga seeds were supplied with 50 μL of extracted root exudates of different rice genotypes. After application, Striga seeds were incubated at 30 °C in the dark for 24 hours. Germinated (seeds with radicle) and non-germinated seeds were counted under a binocular microscope to calculate germination rate (%) by using SeedQuant software (Braguy et al., 2021).

RNA library preparation and transcriptomic analysis

Total rice root RNA was extracted with TRIzol™ (Invitrogen, https://www.thermofisher.com/de/de/home.htmL) using a Direct-zol RNA Miniprep Plus Kit following the manufacturer’s instructions (ZYMO RESEARCH; USA). RNA quality was checked with a Agilent 2100 Bioanalyzer, and RNA concentration was measured using a Qubit 3.0 Fluorometer. The cDNA libraries were constructed following standard protocols and paired-end sequenced on an Illumina NextSeq Sequencer (Illumina HiSeq 4000) by Novogene Bioinformatics Technology Co., Ltd. Total reads were mapped to the rice transcripts using HISAT2 (Kim et al., 2019). Differential gene expression was examined using DESeq2 and established by false discovery rate (FDR) ≤ 0.05 (Love et al., 2014).

Plant material and growth conditions for R. irregularis root colonization

Seed of wild type (cultivar Nipponbare) and independent lines of four Osmax1 - rice mutants (Os900, Os900/1400, Os1400-12SIII and -12SIV) were germinated in pots containing sand and incubated for ten days in a growth chamber under a 14-h light (23 °C)/10-h dark (21 °C). Plants were inoculated with ∼1000 sterile spores of Rhizophagus irregularis DAOM 197198 (Agronutrition, Labège, France). Plants were grown in sterile quartz sand in a growth chamber with the same regime described before and watered with a modified Long-Ashton (LA) solution containing 3.2 μM Na2HPO4·12H2O.

Mycorrhizal roots were collected at two-time points: 10 days post inoculation (dpi), and 40 dpi corresponding to the early and later stages of the mycorrhization process. For the molecular analyses, roots were immediately frozen in liquid nitrogen and stored at −80 °C. At the last time point (40 dpi), mycorrhizal roots were stained with cotton blue (0.1% in lactic acid), and the mycorrhizal colonization level was determined according to Trouvelot et al. (Trouvelot et al., 1986).

Transcript analysis of mycorrhizal plants

Total RNA was extracted from rice roots using the Qiagen Plant RNeasy Kit according to the manufacturer’s instructions (Qiagen, Hilden; Germany). Following the producer’s instructions, samples were treated with TURBO™ DNase (ThermoFischer). The RNA samples were routinely checked for DNA contamination through PCR analysis. Single-strand cDNA was synthesized from 1 μg of total RNA using Super-Script II (Invitrogen), according to the instructions in the user manual. Quantitative RT-PCR (qRT-PCR) was performed using a Rotor-Gene Q 5plex HRM Platform (Qiagen). Each reaction was carried out in a total volume of 15 μL containing 2 μL of diluted cDNA (about 10 ng), 7.5 μl of 2× SYBR Green Reaction Mix, and 2.75 μl of each primer (3 μM). The following PCR program was used: 95°C for 90 s, 40 cycles of 95°C for 15 s, and 60°C for 30 s. A melting curve (80 steps with a heating rate of 0.5°C per 10 s and a continuous fluorescence measurement) was recorded at the end of each run to exclude the generation of non-specific PCR products.

All reactions were performed on at least three biological and two technical replicates. Baseline range and take-off values were automatically calculated using Rotor-Gene Q 5plex software.

The transcript level of OsPt11 (an AM marker gene) was normalized using the OsRubQ1 housekeeping gene (Güimil et al., 2005). Only take-off values leading to a mean with a standard deviation below 0.5 were considered. Statistical elaborations were performed using PAST statistical (version 4) (Hammer et al., 2001).

Ethynyl deoxyuridine (EdU) staining for cell proliferation analysis

For the EdU staining, the seedlings were transferred to 50 ml falcon tubes containing 2 μM 5-ethynyl-2’-deoxyuridine (EdU) in dH2O, so that the roots were completely submerged in the solution, and kept there for 2 hr. The EdU staining was performed as decribed previously, using the Click-iT EdU Alexa Fluor 647 Imaging Kit (Invitrogen, ThermoFisher scientific, USA) (Kirschner et al., 2017). The root were cleared in CLEARSEE clearing solution (Kurihara et al., 2015) at 4 °C in darkness for two weeks, and cell walls were counterstained with 0.1 % Calcofluor White M2R in CLEARSEE overnight in darkness. After washing the roots in CLEARSEE, they were imaged using an inverted confocal microscope (LSM 710, Zeiss) and a 20x objective. Calcofluor White was excited with 405 nm and detected in a detection range of 410 -585 nm. Alexa 647 was excited with 633 nm and detected in a detection range of 638 – 755 nm.

Statistical analysis

Data are represented as mean and their variations as standard deviation. The statistical significance was determined by one-way analysis of variance (one-way ANOVA) and Tukey’s multiple comparison test, using a probability level of p<0.05. All statistical elaborations were performed using GraphPad Prism 9.

Data availability

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. RNA-Seq data can be accessed at NCBI’s Gene Expression Omnibus (GEO) via accession number (GSE221837).

Author Contributions

S.A.-B. and J.Y.W. proposed the concept. J.Y.W and G.-T. E. C. designed the experiments. J.B. generated transgenic Osmax1 mutants. J.B. and G.-T. E. C. conducted genotyping. C.V., V.F., and L.L. investigated mycorrhization studies. J.Y.W. and G.-T. E. C. performed LC-MS analysis as well as RNAseq sample preparation and data analysis. G.-T. E. C., G.K.K., and I.B. prepared and performed cellular level analysis. M.J., J.Y.W., and G.-T. E. C. conducted Striga bioassays. G.-T. E. C., J.Y.W., J.B., M.J., L.B., and A.B. conducted phenotyping experiments. G.-T. E. C., J.Y.W., I.B., L.L., and S. A.-B. analyzed and discussed the data. G.-T. E. C., J.Y.W., J.B., and S. A.-B. wrote the original manuscript. All authors read, edited, and approved the manuscript.

Competing interests

The authors declare no competing interests.

Acknowledgments

We sincerely thank the support from the members of KAUST Analytical Core Lab and the Bioactives lab. We thank Dr. Abdel Gabbar Babiker for providing Striga hermonthica seeds and Prof. Tadao Asami for providing TIS108. This work was supported by baseline funding given to S. A-B from King Abdullah University of Science and Technology (KAUST) and the Bill and Melinda Gates Foundation (grant number OPP1136424).

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Supplementary Material

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Disruption of the rice 4-DEOXYOROBANCHOL HYDROXYLASE unravels specific functions of canonical strigolactones
Guan-Ting Erica Chen, Jian You Wang, Cristina Votta, Justine Braguy, Muhammad Jamil, Gwendolyn K Kirschner, Valentina Fiorilli, Lamis Berqdar, Aparna Balakrishna, Ikram Blilou, Luisa Lanfranco, Salim Al-Babili
bioRxiv 2023.04.03.535333; doi: https://doi.org/10.1101/2023.04.03.535333
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