Biologically Relevant Methods to Test How Microbes Colonize Maize Styles (Silks): Case Study of a Pantoea Strain

Maize silks (style) receive wind-transmitted pollen. Thereafter, male gametes travel through the silks to reach ovules. Pollinated silks contain a microbiome, members of which are predicted to promote host reproduction and abiotic/biotic stress tolerance during fertilization. It is unknown whether pollinated silk bacteria originate environmentally (air/pollen) or from maternal tissue. Methods are lacking to test microbial colonization of silks in their native habitat [on intact maize inflorescences (cobs) encased by husk leaves]. Current methods focus on naked silks attached to dehusked cob pieces. Here, two novel methods are presented to enable research on silk microbes in their native habitat. Method 1 tests whether silk-associated bacteria with potential environmental origins are attracted toward ovules. Method 2 distinguishes whether a microbe colonizes silks from the environment or maternal parent. Biosafety containment was enabled by housing microbe-treated cobs in large jars. Using these methods, a model bacterial isolate from fertilization-stage silks (DsRed-tagged Pantoea-E04) was shown to colonize husk-covered silks after inoculating exposed silk tips; E04 could not colonize from the cob base, suggesting an environmental origin. In support, E04 colonized silks more frequently when cobs were uncut and oriented vertically. These protocols will enable more biologically relevant investigation of silk microbiomes and pathogens.