CRISPR/Cas9‐mediated single and biallelic knockout of poplar <i>STERILE APETALA</i> (<i>PopSAP</i>) leads to complete reproductive sterility

Abstract

The spread of highly domesticated, exotic, or genetically modified organisms into wild and feral populations and/or their genes beyond the boundaries of plantations can negatively, irreversibly impact the health of native species and pose significant management problems in human-dominated ecosystems (Snow et al., 2005). Therefore, it is imperative to minimize or, if possible, completely avert the risks associated with such spread. Containment refers to approaches used to prevent the spread of such organisms (Brunner et al., 2007). Containment approaches were first inspired in the early 1990s by the demonstration of genetically engineered male and female sterility (Mariani et al., 1990). Reproductive sterility effectively minimizes or voids any risk of spreading genes/transgenes into the wild by means of sexual reproduction by significantly reducing or preventing the generation of gametes. Inducing floral sterility is a feasible and relevant approach, particularly in trees. Many major tree crops, including poplar, are clonally propagated with rooted cuttings, and thus, a lack of seeds does not interfere with their propagation. Delaying or eliminating flowering is beneficial since this channels more assimilates to vegetative growth, the primary product in the forest plantation (Brunner et al., 2007). Two major approaches to alter sexual reproduction have been tested. The first, known as floral ablation, involves the expression of toxic genes under a floral-predominant promoter. The expression of toxins in floral tissues either renders these tissues non-functional or destroys them. A major problem with floral ablation has been the ‘leaky’ expression of the toxic genes outside flower tissues, which significantly compromises vegetative growth (Wei et al., 2006). The second approach, which we employ in this study, involves the modification of genes that affect normal reproductive development or interfere with the transition to reproductive growth. STERILE APETALA (SAP) was first discovered in Arabidopsis thaliana as the result of a SAP loss-of-function mutation that caused complete male and female sterility (Byzova et al., 1999). More recently, work in A. thaliana and our work in poplar showed that, in addition to having a role in reproductive development, SAP is also involved in vegetative growth, particularly in the regulation of leaf size (Li et al., 2018; Yordanov et al., 2017). Because of the similar roles of SAP in the regulation of vegetative growth in A. thaliana and poplar, we hypothesized that poplar SAP (PopSAP) knockout will lead to sterility in poplar as in A. thaliana. Although long generation cycles are one of the greatest impediments to testing sterility technologies in poplar, this can be overcome by using inducible expression of FT (FLOWERING LOCUS T) orthologs (Zhang et al., 2010). First, we improved this inducible system to make it more suitable for testing sterility approaches in poplar (Azeez and Busov, 2019). After determining that the inconsistent, slow flowering was caused by low levels of FT induction brought about by the heat inductive system, we developed a more efficient method employing repeated cycles of heat induction followed by resting periods for recovery from heat shock (Azeez and Busov, 2019). This method was successfully tested in both male (353-FT, Populus tremula x tremuloides) and female (717-FT, Populus tremula x alba) poplar genotypes. Next, we utilized the modified induction method to test the efficiency of PopSAP knockout as a sterility technology. CRISPR/Cas9 was used to knockout PopSAP in both the male (353-FT) and female (717-FT) genotypes. The target site was selected based on location in the gene and GC content using Aspen DB (http://aspendb.uga.edu/index.php/databases/spta-717-genome; Figure 1a). The specificity of the sgRNA was also checked by BLAST searches in the aspen (http://popgenie.org/) and 717 genome databases (http://aspendb.uga.edu/index.php/databases/spta-717-genome). The expression cassette was assembled in a shuttle vector containing the A. thaliana AtU3d promoter and then transferred into the pYLCRISPR/Cas9Pubi-H binary vector (Ma et al., 2015). The construct was sequence-verified, transformed into Agrobacterium strain C58, and finally transformed into the male (353-FT) and female (717-FT) genotypes using leaf tissues. We produced numerous transgenic plants and sequenced more than 20 independent lines to identify knockouts of the PopSAP gene. We identified 10 lines with frameshift indels in one allele (Figure 1b, c) in both male and female genotypes but only one independent line (18-1/A) in the female genotype carrying frameshift mutations in both alleles (Figure 1b). This single line had significantly impaired growth (Figure 1d, f), suggesting that biallelic loss-of-function mutations in the PopSAP gene are difficult to recover likely due to impaired growth, problems with regeneration or combinations of these. Since PopSAP is a single gene in poplar and other genomes (Yordanov et al., 2017), there are no redundant copies of the gene; therefore, knocking out PopSAP likely significantly impairs growth and development. For this reason, we proceeded to characterize plants carrying heterozygous knockouts (only one allele mutated) and the single line carrying the biallelic mutations. We selected six independent lines carrying different PopSAP knockout mutations for further analysis, including three heterozygous lines in the male (353-FT) genotype, two heterozygous lines in the female (717-FT) genotype and the single female line with biallelic homozygous mutations (Line18-1/A). We then used our improved flower induction system to test the effect of these mutations on flower initiation. In contrast to control plants, the heterozygous knockouts in both male and female genotypes caused complete sterility with no initiation of inflorescences; this was also true for the biallelic knockout in the female genotype (Figure 1d-g). These results differ from A. thaliana, where flowers were observed but were sterile (Byzova et al., 1999). These differences are likely attributable to key differences in inflorescence development in the two species. Poplar initiates inflorescence development nearly a year before they fully develop, but in A. thaliana this occurs within the same year. In A. thaliana and poplar (Yordanov et al., 2017), SAP regulates vegetative growth and development. For this reason, we determined whether the mutations in PopSAP affect vegetative growth. Indeed, in the female 717-FT genotype, the only plant carrying biallelic PopSAP loss-of-function mutations was significantly shorter than control plants (Figure 1d, f). In the male 353-FT genotype, some heterozygous PopSAP knockouts showed an appreciable reduction in growth (height), though this was not statistically significant (Figure 1e, g). Therefore, while heterozygous and biallelic knockouts of PopSAP likely affect vegetative growth, these effects differ depending on the clone used as well as heterozygous vs. biallelic knockout. Long-term field testing is necessary to more reliably assess the effect of observed changes in vegetative growth. Studies in A. thaliana are beginning to clarify the role of SAP in vegetative growth. These studies demonstrate that by modifying SAP target genes it is possible to mitigate the effects of SAP knockout on vegetative growth; additional studies are necessary to ensure that sterility phenotypes are not compromised under these conditions. Our results indicate that SAP is a promising target for developing sterility-based containment technologies in poplar. Further testing is needed, particularly under field conditions, to validate the robustness of the system and its effects on biomass accumulation. This project was supported by Biotechnology Risk Assessment Grant Program Competitive Grant no. 2016-33522-25626 from the U.S.D.A. We thank Galina Agapova and Naomi Ojala for propagation and care of the transgenic lines. The authors declare no financial conflict of interest. V.B. conceived the research. A.A. designed and executed the experiments. Both authors wrote the article.

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