**4. Materials and Methods**

### *4.1. CRISPR*/*Cas9 Vector Construction and Rice Transformation*

To generate *OsITPK6* mutants, the 1st exon of *OsITPK6* (*Os09g0518700*) was chosen as a target (Figure 1A). The sgRNAs were designed by searching UniProt for precise positions (http://www.uniprot. org/), and CRISPR-P program (http://cbi.hzau.edu.cn/cgi-bin/CRISPR/) was used to minimize off-target effects [37]. Because of the homology of the target sequence among *OsITPK* genes, it is unlikely to cause mutations in the other five *OsITPK* homolog genes (Figure S2). DNA oligonucleotides were synthesized (Tsingke, Hangzhou, China) for the construction of a CRISPR/Cas9 vector, pH\_itpk6, using the pHun4c12s as the starting plasmid, which harbors a *CYP81A6*-hpRNAi element [38] and

was modified from pHun4c12 [39]. Correspondingly, the pH\_itpk6 plasmid was transformed into *Agrobacterium tumefaciens* and used for rice transformation.

Rice calli were induced from mature seeds of the cultivar 'Xidao 1' (*O. sativa* L. *japonica*) and were transformed with the pH-itpk6 vector by *Agrobacterium*-mediated transformation according to reference [40]. Transgenic plantlets were regenerated from hygromycin-resistant calli and acclimatized inside a moist growth chamber (28 ◦C with a 12 h photoperiod) for one week before being transplanted to experimental facilities.

Ethics Approval and Consent to Participate: The experiments did not involve endangered or protected species. No specific permits were required for these locations/activities.
