Novel disease resistance gene paralogs created by CRISPR/Cas9 in soy.

KEY MESSAGE: Novel disease resistance gene paralogues are generated by targeted chromosome cleavage of tandem duplicated NBS-LRR gene complexes and subsequent DNA repair in soybean. This study demonstrates accelerated diversification of innate immunity of plants using CRISPR. Nucleotide-binding-site-leucine-rich-repeat (NBS-LRR) gene families are key components of effector-triggered immunity. They are often arranged in tandem duplicated arrays in the genome, a configuration that is conducive to recombinations that will lead to new, chimeric genes. These rearrangements have been recognized as major sources of novel disease resistance phenotypes. Targeted chromosome cleavage by CRISPR/Cas9 can conceivably induce rearrangements and thus emergence of new resistance gene paralogues. Two NBS-LRR families of soy have been selected to demonstrate this concept: a four-copy family in the Rpp1 region (Rpp1L) and a large, complex locus, Rps1 with 22 copies. Copy-number variations suggesting large-scale, CRISPR/Cas9-mediated chromosome rearrangements in the Rpp1L and Rps1 complexes were detected in up to 58.8% of progenies of primary transformants using droplet-digital PCR. Sequencing confirmed development of novel, chimeric paralogs with intact open reading frames. These novel paralogs may confer new disease resistance specificities. This method to diversify innate immunity of plants by genome editing is readily applicable to other disease resistance genes or other repetitive loci.

Azteca ants maintain unique microbiomes across functionally distinct nest chambers.

The microbiome of built structures has considerable influence over an inhabitant's well-being, yet the vast majority of research has focused on human-built structures. Ants are well-known architects, capable of constructing elaborate dwellings, the microbiome of which is underexplored. Here, we explore the bacterial and fungal microbiomes in functionally distinct chambers within and outside the nests of Azteca alfari ants in Cecropia peltata trees. We predicted that A. alfari colonies (1) maintain distinct microbiomes within their nests compared to the surrounding environment, (2) maintain distinct microbiomes among nest chambers used for different functions, and (3) limit both ant and plant pathogens inside their nests. In support of these predictions, we found that internal and external nest sampling locations had distinct microbial communities, and A. alfari maintained lower bacterial richness in their 'nurseries'. While putative animal pathogens were suppressed in chambers that ants actively inhabited, putative plant pathogens were not, which does not support our hypothesis that A. alfari defends its host trees against microbial antagonists. Our results show that ants influence microbial communities inside their nests similar to studies of human homes. Unlike humans, ants limit the bacteria in their nurseries and potentially prevent the build-up of insect-infecting pathogens. These results highlight the importance of documenting how indoor microbiomes differ among species, which might improve our understanding of how to promote indoor health in human dwellings.

Symbiosis in the Soil: Citizen Microbiology in Middle and High School Classrooms.

Microorganisms are vital to environmental health, yet their association with disease often overshadows these benefits. Building citizen-science activities around the positive role of microorganisms and an understanding of their ubiquity can begin to dispel misconceptions while simultaneously engaging the public in research. Here, we describe a citizen-science microbiology project geared toward implementation in middle and high school classrooms. Students culture environmental microorganisms and document microbial diversity of plant root systems compared with adjacent bulk soil. Results contribute data toward research on microbiome recruitment of weeds and other successful plants while addressing core topics in science education.

Slowing PCR ramp speed reduces chimera formation from environmental samples.

Chimeric sequences falsely increase the apparent diversity within samples. To examine chimera formation in PCR products from environmental DNA, clone libraries were prepared using different ramp speeds to reach the designated temperatures for each step of the PCR program. Slowing the thermocycler ramp speed to 1 °C s(-1) reduced chimera formation.