GFP-labeled pathogens have been used to study the systematic colonization and infection of Fusarium spp. in maize [20] and [21]. Red fluorescent protein (DsRed), discovered in radiating mushroom coral (Discosoma striata), has an emission spectrum in the far-red zone [22] and permits dual or multi-color labeling of many fungal species. The DsRed protein has been used effectively to label a number

of filamentous fungi, S3I-201 order such as Aspergillus, Trichoderma, and Oculimacular spp. [23], [24] and [25]. In a previous study, we generated F. verticillioides strains expressing red fluorescence by introducing the gene DsRed via Agrobacterium tumefaciens-mediated transformation (ATMT) [26]. Using a DsRed-labeled fungal strain, this study was initiated to investigate the differences

in colonization Neratinib in vitro and reaction of resistant and susceptible maize lines challenged with F. verticillioides. Wild type strain Fv-1 of F. verticillioides was isolated from Yayuan County, Jilin Province, China. Its identity was confirmed by morphological and interval transcribed spacer (ITS) sequence analyses. Susceptible maize inbred lines B73, P138 and Lu 9804, and the resistant lines Qi 319, Dan 340 and Zhongzi 01, were used in the study. The plasmid pCAMDsRed [27], which contains the gene DsRed driven by the promoter PgpdA, as well the selectable gene hpg for resistance to the antibiotic hygromycin, was used in ATMT of F. verticillioides as described previously [26] and [28]. Analyses of mitotic stability of DsRed protein expression,

growth rates of colonies, and metabolism of extracellular enzymes (i.e., protease, Resminostat cellulase, amylase, and pectase) in the transformants were performed to characterize the DsRed-labeled strain of F. verticillioides [26]. Seeds of the maize inbred lines were washed with running water, surface sterilized in 75% alcohol for 5 min and in 0.4% sodium hypochlorite for 15–20 min, and then rinsed with distilled water. The surface-sterilized seeds were sown in pots (10 L) filled with vermiculite in a greenhouse set at 25–30 °C for 16 h of light and at 16 °C for 8 h of darkness. When the second seedling leaves were unfolded, the top 12 cm of vermiculite was removed from pots, mixed with the suspensions of the DsRed-labeled fungus (108 conidia mL− 1) at a rate of 1:5 (V/W), and returned to the pots. In the untreated checks, soil similarly treated with distilled water was used as mock inoculation. Root cross sections were prepared using a Microtome (MTH-I, Tokyo, Japan) without fixation to ensure living root cells and real-time observation. Systemic colonization by F. verticillioides in root tissues was determined by observing the red fluorescence emitted by the DsRed-labeled fungus with an epifluorescent microscope (BX60, Olympus, Tokyo, Japan) under emission wavelengths of 515/560 nm. Light microscopy was performed with the same microscope without a filter. To determine the infection and colonization by F.