Genome Editing-Based Engineering of CESA3 Dual Cellulose-Inhibitor-Resistant Plants

Abstract

The rapid appearance of herbicide-resistant weeds combined with a lack of novel herbicides being brought to market reduces crop production, thereby threatening food security worldwide. Here, we report on the use of the previously identified cellulose biosynthesis-inhibiting chemical compound C17 as a potential herbicide. Toxicity tests showed that C17 efficiently inhibits the growth of various weeds and widely cultivated dicotyledonous crops, whereas only slight or no growth inhibition was observed for monocotyledonous crops. Surprisingly, when exposed to a mixture of C17 and one of two well-known cellulose biosynthesis inhibitors (CBIs), isoxaben and indaziflam, an additive growth inhibition was observed, demonstrating that C17 has a different mode of action that can be used to sensitize plants toward known CBIs. Moreover, we demonstrate that a C17-resistant CESA3 allele can be used as a positive transformation selection marker and that C17 resistance can be obtained through genome engineering of the wild-type CESA3 allele using clustered regularly interspaced short palindromic repeats-mediated base editing. This editing system allowed us to engineer C17 tolerance in an isoxaben-resistant line, resulting in double herbicideresistant plants.

Endosidin20 is a broad-spectrum cellulose synthesis inhibitor with an herbicidal function

“…The previous Quinoxyphen and isoxaben dual tolerant CESA1 and CESA3 double mutant cesa1 aegeus / cesa3 ixr1-2 also showed a far more pronounced dwarf phenotype than either of the single mutants (Harris et al, 2012). However, recently reported CESA3 S983F and CESA6 ixr2-1 double mutant shows isoxaben and C17 dual tolerance but does not seem to have obvious growth phenotypes (Hu et al, 2019), which indicates different combination of mutated CESAs may affect the plant growth differently and it is still possible to obtain CESA dual drug tolerant plant without growth penalty. It is worth trying to create double amino acids mutations in CESA6 for irx2-1 ; es20r1 and test for the plants’ response to both ES20 and isoxaben and examine their growth phenotypes.…”

“…Interestingly, the mutations at different CESAs, especially the primary cell wall related CESA 1, 3 and 6, have been found to cause reduced sensitivity to CBIs (Scheible et al, 2001; Desprez et al, 2002; Tateno et al, 2016; Hu et al, 2018). The CBIs and the reduced sensitivity mutants are valuable resources for the development of novel herbicides and for the breeding of herbicide tolerant crops which can be accomplished by gene editing technology (Hu et al, 2019).…”

“…ES20 tolerance caused by single amino acid change in CESA6 indicates that it is possible to create other ES20-tolerant crop species using gene editing technology. CRISPR-mediated gene editing has been suggested to obtain C17-tolerant plants (Hu et al, 2019) and it is possible to create ES20 tolerant plants as well with CRISPR technology. We also show that it is possible to create plants that have dual tolerance to ES20 and isoxaben.…”

“…Interestingly, the inhibitor of mitochondrial complex III can also reduce plants’ sensitivity to C17 treatment, indicating that C17 might have a complex mode of action instead of directly targets CESA to inhibit plant growth. C17 has inhibitory effect on broad plant species, indicating it might be a good candidate for herbicide development (Hu et al, 2019). Indaziflam is a potent CBI that has been commercialized as an herbicide (Brabham et al, 2014).…”

Comparative transcriptome analysis revealed the cooperative regulation of sucrose and IAA on adventitious root formation in lotus (Nelumbo nucifera Gaertn)

“…Finally, the AR primordium continues to develop until the AR break through the epidermis of the stem or leaf (the maintenance phase) [10]. Various internal (gene regulation or expression) and external factors (temperature, light, mechanical damage, hormone) are involved in the morphological and anatomical structure formation, physiological and biochemical or molecular regulation of the organ (including root) development [9,[11][12][13]. Therefore, the formation of AR is a heritable quantitative trait.…”