Novel Mutant Alleles Reveal a Role of the Extra-Large G Protein in Rice Grain Filling, Panicle Architecture, Plant Growth, and Disease Resistance

Abstract

Plant growth and grain filling are the key agronomical traits for grain weight and yield of rice. The continuous improvement in rice yield is required for a future sustainable global economy and food security. The heterotrimeric G protein complex containing a canonical α subunit (RGA1) couples extracellular signals perceived by receptors to modulate cell function including plant development and grain weight. We hypothesized that, besides RGA1, three atypical, extra-large GTP-binding protein (XLG) subunits also regulate panicle architecture, plant growth, development, grain weight, and disease resistance. Here, we identified a role of XLGs in agronomic traits and stress tolerance by genetically ablating all three rice XLGs individually and in combination using the CRISPR/Cas9 genome editing in rice. For this study, eight (three single, two double, and three triple) null mutants were selected. Three XLG proteins combinatorically regulate seed filling, because loss confers a decrease in grain weight from 14% with loss of one XLG and loss of three to 32% decrease in grain weight. Null mutations in XLG2 and XLG4 increase grain size. The mutants showed significantly reduced panicle length and number per plant including lesser number of grains per panicle compared to the controls. Loss-of-function of all individual XLGs contributed to 9% more aerial biomass compared to wild type (WT). The double mutant showed improved salinity tolerance. Moreover, loss of the XLG gene family confers hypersensitivity to pathogens. Our findings suggest that the non-canonical XLGs play important roles in regulating rice plant growth, grain filling, panicle phenotype, stress tolerance, and disease resistance. Genetic manipulation of XLGs has the potential to improve agronomic properties in rice.

An efficient transformation method for genome editing of elite bread wheat cultivars

“…This vector system resulted in nearly 90% transformation efficiency for single gene editing in rice. Earlier, we successfully generated triple edited Osxlg mutants using this vector system ( Biswal et al., 2022 ). The duplex guide oligo was inserted into the BsaI site of pRGEB32 using InFusion ® cloning at GenScript Biotech Inc., USA ( Figure 1C ).…”

“…Target sequences were amplified by Phusion DNA polymerase (New England Biolabls, United States) using gene-specific primers flanking the target site ( Supplementary Table S3 ) using the protocol mentioned by Biswal et al. (2022) .…”

Bioinformatic and literature assessment of toxicity and allergenicity of a CRISPR-Cas9 engineered gene drive to control the human malaria mosquito vector Anopheles gambiae

“…Cas9 has commonly been used in studies to evaluate allergens and immune-related diseases such as egg allergies [147], or to remove allergens from products such as soybean through site-directed mutagenesis [148] or target genes involved in allergic rhinitis [149]; or remove and functionally evaluate other allergens [150][151][152][153][154][155], with no literature studies having implicated Cas9 proteins themselves in allergenicity. Importantly, Nakajima et al [156] reported that Cas9 was subject to rapid digestion in vitro and unlikely to cause food allergy in the case of accidental ingestion.…”

Cytological, transcriptome and miRNome temporal landscapes decode enhancement of rice grain size

“…GS regulation in rice is extremely diverse with involvement of genes controlling hormones [ 15 ], G-protein signaling [ 16 – 18 ], ubiquitin proteasome pathway [ 19 , 20 ], and starch metabolism [ 21 ]. They can also be transcriptional activators [ 11 ], microtubule-associated proteins [ 22 ], chromatin and histone modifiers [ 23 ], or sucrose transporters [ 24 ].…”