| Developing maize kernels are vulnerable to colonization by microbes. When colonization allows proliferation of the microbe at the expense of the host, disease occurs. The ascomycete fungal pathogens Aspergillus flavus and Fusarium verticillioides are capable of colonizing maize kernels, causing ear rots and contamination of the kernel with mycotoxins. These diseases lead to significant losses of crop yield and quality, and constitute a threat to food safety and human health. Thus, the significance of these diseases has prompted extensive research efforts to understand these plant-parasite interactions. However, pathogenesis and resistance mechanisms remain poorly characterized, hampering the development of effective control strategies. No commercial maize lines are completely resistant to these fungi. We applied an integrated approach consisting of histology, in situ gene expression and transcriptional profiling to better understand the nature of the interactions that occur between maize kernels and these fungi. Maize inbred line B73 was hand pollinated and inoculated with either A. flavus or F. verticillioides by wounding the kernel with a needle bearing conidia. Histological staining of the kernel sections revealed fungal mycelium in kernels adjacent to the inoculation site by 48 hours post inoculation (hpi), and in all tissues at 96 hpi. Compared with F. verticillioides, A. flavus more aggressively colonized kernel tissue and formed a unique biofilm-like structure around the scutellum. Transcriptome profiling using RNA-sequencing (RNA-seq) coupled with pathway analysis showed that these fungi were recognized by the kernel tissues prior to visible colonization. Infection of the kernel by these fungi induced transcriptional changes in defense-related genes, hormone signaling networks, as well as primary and secondary metabolism pathways. To dissect tissue-specific responses of the kernel, RNA in situ hybridization and histological staining were carried out in adjacent serial sections. We found that two maize genes, pathogenesis related protein, maize seeds (PRms) and shrunken-1 (Sh1) , were expressed in the aleurone and scutellum during infection by these fungi. By staining the adjacent sections, we found that these genes were induced in the tissue before the establishment of fungal colonization. Integration of histology, in situ gene expression and transcriptional profiling to study pathogenesis of maize kernels by these two fungi revealed distinctive and common features between the two pathosystems, and provided information that will facilitate the development of resistance genotypes in maize. |
