Spatially and temporally distinct Ca2+ changes in Lotus japonicus roots orient fungal-triggered signalling pathways towards symbiosis or immunity.

Plants activate an immune or symbiotic response depending on the detection of distinct signals from root-interacting microbes. Both signalling cascades involve Ca2+ as a central mediator of early signal transduction. In this study, we combined aequorin- and cameleon-based methods to dissect the changes in cytosolic and nuclear Ca2+ concentration caused by different chitin-derived fungal elicitors in Lotus japonicus roots. Our quantitative analyses highlighted the dual character of the evoked Ca2+ responses taking advantage of the comparison between different genetic backgrounds: an initial Ca2+ influx, dependent on the LysM receptor CERK6 and independent of the common symbiotic signalling pathway (CSSP), is followed by a second CSSP-dependent and CERK6-independent phase, that corresponds to the well-known perinuclear/nuclear Ca2+ spiking. We show that the expression of immunity marker genes correlates with the amplitude of the first Ca2+ change, depends on elicitor concentration, and is controlled by Ca2+ storage in the vacuole. Our findings provide an insight into the Ca2+-mediated signalling mechanisms discriminating plant immunity- and symbiosis-related pathways in the context of their simultaneous activation by single fungal elicitors.

Quantifying root colonization by a symbiotic fungus using automated image segmentation and machine learning approaches.

Arbuscular mycorrhizas (AM) are one of the most widespread symbiosis on earth. This plant-fungus interaction involves around 72% of plant species, including most crops. AM symbiosis improves plant nutrition and tolerance to biotic and abiotic stresses. The fungus, in turn, receives carbon compounds derived from the plant photosynthetic process, such as sugars and lipids. Most studies investigating AM and their applications in agriculture requires a precise quantification of the intensity of plant colonization. At present, the majority of researchers in the field base AM quantification analyses on manual visual methods, prone to operator errors and limited reproducibility. Here we propose a novel semi-automated approach to quantify AM fungal root colonization based on digital image analysis comparing three methods: (i) manual quantification (ii) image thresholding, (iii) machine learning. We recognize machine learning as a very promising tool for accelerating, simplifying and standardizing critical steps in analysing AM quantification, answering to an urgent need by the scientific community studying this symbiosis.

Size matters: three methods for estimating nuclear size in mycorrhizal roots of Medicago truncatula by image analysis.

BACKGROUND: The intracellular accommodation of arbuscular mycorrhizal (AM) fungi involves a profound molecular reprogramming of the host cell architecture and metabolism, based on the activation of a symbiotic signaling pathway. In analogy with other plant biotrophs, AM fungi are reported to trigger cell cycle reactivation in their host tissues, possibly in support of the enhanced metabolic demand required for the symbiosis. RESULTS: We here compare the efficiency of three Fiji/ImageJ image analysis plugins in localizing and quantifying the increase in nuclear size - a hallmark of recursive events of endoreduplication - in M. truncatula roots colonized by the AM fungus Gigaspora margarita. All three approaches proved to be versatile and upgradeable, allowing the investigation of nuclear changes in a complex tissue; 3D Object Counter provided more detailed information than both TrackMate and Round Surface Detector plugins. On this base we challenged 3D Object Counter with two case studies: verifying the lack of endoreduplication-triggering responses in Medicago truncatula mutants with a known non-symbiotic phenotype; and analysing the correlation in space and time between the induction of cortical cell division and endoreduplication upon AM colonization. Both case studies revealed important biological aspects. Mutant phenotype analyses have demonstrated that the knock-out mutation of different key genes in the symbiotic signaling pathway block AM-associated endoreduplication. Furthermore, our data show that cell divisions occur during initial stages of root colonization and are followed by recursive activation of the endocycle in preparation for arbuscule accommodation. CONCLUSIONS: In conclusion, our results indicate 3D Object Counter as the best performing Fiji/ImageJ image analysis script in plant root thick sections and its application highlighted endoreduplication as a major feature of the AM pre-penetration response in root cortical cells.

Local endoreduplication as a feature of intracellular fungal accommodation in arbuscular mycorrhizas.

The intracellular accommodation of arbuscular mycorrhizal (AM) fungi is a paradigmatic feature of this plant symbiosis that depends on the activation of a dedicated signaling pathway and the extensive reprogramming of host cells, including striking changes in nuclear size and transcriptional activity. By combining targeted sampling of early root colonization sites, detailed confocal imaging, flow cytometry and gene expression analyses, we demonstrate that local, recursive events of endoreduplication are triggered in the Medicago truncatula root cortex during AM colonization. AM colonization induces an increase in ploidy levels and the activation of endocycle specific markers. This response anticipates the progression of fungal colonization and is limited to arbusculated and neighboring cells in the cortical tissue. Furthermore, endoreduplication is not induced in M. truncatula mutants for symbiotic signaling pathway genes. On this basis, we propose endoreduplication as part of the host cell prepenetration responses that anticipate AM fungal accommodation in the root cortex.