CRISPR/Cas9 Based Cell-Type Specific Gene Knock-Out in Arabidopsis Roots.

CRISPR/Cas9 (hereafter Cas9)-mediated gene knockout is one of the most important tools for studying gene function. However, many genes in plants play distinct roles in different cell types. Engineering the currently used Cas9 system to achieve cell-type-specific knockout of functional genes is useful for addressing the cell-specific functions of genes. Here we employed the cell-specific promoters of the WUSCHEL RELATED HOMEOBOX 5 (WOX5), CYCLIND6;1 (CYCD6;1), and ENDODERMIS7 (EN7) genes to drive the Cas9 element, allowing tissue-specific targeting of the genes of interest. We designed the reporters to verify the tissue-specific gene knockout in vivo. Our observation of the developmental phenotypes provides strong evidence for the involvement of SCARECROW (SCR) and GIBBERELLIC ACID INSENSITIVE (GAI) in the development of quiescent center (QC) and endodermal cells. This system overcomes the limitations of traditional plant mutagenesis techniques, which often result in embryonic lethality or pleiotropic phenotypes. By allowing cell-type-specific manipulation, this system has great potential to help us better understand the spatiotemporal functions of genes during plant development.

Brassinosteroid signaling restricts root lignification by antagonizing SHORT-ROOT function in Arabidopsis.

Cell wall lignification is a key step in forming functional endodermis and protoxylem (PX) in plant roots. Lignified casparian strips (CS) in endodermis and tracheary elements of PX are essential for selective absorption and transport of water and nutrients. Although multiple key regulators of CS and PX have been identified, the spatial information that drives the developmental shift to root lignification remains unknown. Here, we found that brassinosteroid (BR) signaling plays a key role in inhibiting root lignification in the root elongation zone. The inhibitory activity of BR signaling occurs partially through the direct binding of BRASSINAZOLE-RESISTANT 1 (BZR1) to SHORT-ROOT (SHR), repressing the SHR-mediated activation of downstream genes that are involved in root lignification. Upon entering the mature root zone, BR signaling declines rapidly, which releases SHR activity and initiates root lignification. Our results provide a mechanistic view of the developmental transition to cell wall lignification in Arabidopsis thaliana roots.

SlSPX1-SlPHR complexes mediate the suppression of arbuscular mycorrhizal symbiosis by phosphate repletion in tomato.

Forming mutualistic symbioses with arbuscular mycorrhizae (AMs) improves the acquisition of mineral nutrients for most terrestrial plants. However, the formation of AM symbiosis usually occurs under phosphate (Pi)-deficient conditions. Here, we identify SlSPX1 (SYG1 (suppressor of yeast GPA1)/Pho81(phosphate 81)/XPR1 (xenotropic and polytropic retrovirus receptor 1) as the major repressor of the AM symbiosis in tomato (Solanum lycopersicum) under phosphate-replete conditions. Loss of SlSPX1 function promotes direct Pi uptake and enhances AM colonization under phosphate-replete conditions. We determine that SlSPX1 integrates Pi signaling and AM symbiosis by directly interacting with a set of arbuscule-induced SlPHR proteins (SlPHR1, SlPHR4, SlPHR10, SlPHR11, and SlPHR12). The association with SlSPX1 represses the ability of SlPHR proteins to activate AM marker genes required for the arbuscular mycorrhizal symbiosis. SlPHR proteins exhibit functional redundancy, and no defective AM symbiosis was detected in the single mutant of SlPHR proteins. However, silencing SlPHR4 in the Slphr1 mutant background led to reduced AM colonization. Therefore, our results support the conclusion that SlSPX1-SlPHRs form a Pi-sensing module to coordinate the AM symbiosis under different Pi-availability conditions.

Symplastic communication in the root cap directs auxin distribution to modulate root development.

Root cap not only protects root meristem, but also detects and transduces the signals of environmental changes to affect root development. The symplastic communication is an important way for plants to transduce signals to coordinate the development and physiology in response to the changing enviroments. However, it is unclear how the symplastic communication between root cap cells affects root growth. Here we exploit an inducible system to specifically block the symplastic communication in the root cap. Transient blockage of plasmodesmata (PD) in differentiated collumella cells severely impairs the root development in Arabidopsis, in particular in the stem cell niche and the proximal meristem. The neighboring stem cell niche is the region that is most sensitive to the disrupted symplastic communication and responds rapidly via the alteration of auxin distribution. In the later stage, the cell division in proximal meristem is inhibited, presumably due to the reduced auxin level in the root cap. Our results reveal the essential role of the differentiated collumella cells in the root cap mediated signaling system that directs root development.

SHORT-ROOT paralogs mediate feedforward regulation of D-type cyclin to promote nodule formation in soybean.

Nitrogen fixation in soybean takes place in root nodules that arise from de novo cell divisions in the root cortex. Although several early nodulin genes have been identified, the mechanism behind the stimulation of cortical cell division during nodulation has not been fully resolved. Here we provide evidence that two paralogs of soybean SHORT-ROOT (GmSHR) play vital roles in soybean nodulation. Expression of GmSHR4 and GmSHR5 (GmSHR4/5) is induced in cortical cells at the beginning of nodulation, when the first cell divisions occur. The expression level of GmSHR4/5 is positively associated with cortical cell division and nodulation. Knockdown of GmSHR5 inhibits cell division in outer cortical layers during nodulation. Knockdown of both paralogs disrupts the cell division throughout the cortex, resulting in poorly organized nodule primordia with delayed vascular tissue formation. GmSHR4/5 function by enhancing cytokinin signaling and activating early nodulin genes. Interestingly, D-type cyclins act downstream of GmSHR4/5, and GmSHR4/5 form a feedforward loop regulating D-type cyclins. Overexpression of D-type cyclins in soybean roots also enhanced nodulation. Collectively, we conclude that the GmSHR4/5-mediated pathway represents a vital module that triggers cytokinin signaling and activates D-type cyclins during nodulation in soybean.