RiCRN1, a Crinkler Effector From the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis, Functions in Arbuscule Development.

Arbuscular mycorrhizal (AM) symbiosis is one of the most prominent and beneficial plant-microbe interactions that facilitates mineral nutrition and confers tolerance to biotic and abiotic stresses. AM fungi colonize the root cortex and develop specialized structures called arbuscules where the nutrient exchange takes place. Arbuscule development is a highly controlled and coordinated process requiring the involvement of many plant proteins recruited at that interface. In contrast, much less is known about the fungal proteins involved in this process. Here, we have identified an AM fungal effector that participates in this developmental step of the symbiosis. RiCRN1 is a crinkler (CRN) effector that belongs to a subfamily of secreted CRN proteins from R. irregularis. CRNs have been so far only functionally characterized in pathogenic microbes and shown to participate in processes controlling plant cell death and immunity. RiCRN1 accumulates during symbiosis establishment parallel to MtPT4, the gene coding for an arbuscule-specific phosphate transporter. Expression in Nicotiana benthamiana leaves and in Medicago truncatula roots suggest that RiCRN1 is not involved in cell death processes. RiCRN1 dimerizes and localizes to nuclear bodies, suggesting that, similar to other CRNs, it functions in the plant nucleus. Downregulation of RiCRN1 using host-induced gene silencing led to an impairment of the symbiosis in M. truncatula and to a reduction of MtPT4, while ectopic expression of RiCRN1, surprisingly, led to a drastic reduction in arbuscule size that correlated with a decrease not only in MtPT4 but also in MtBCP1, a marker for initial stages of arbuscule development. Altogether, our results suggest that a tightly regulated expression in time and space of RiCRN1 is critical for symbiosis progression and for the proper initiation of arbuscule development.

Symbiotic Fungi Control Plant Root Cortex Development through the Novel GRAS Transcription Factor MIG1.

In an approaching scenario of soil nutrient depletion, root association with soil microorganisms can be key for plant health and sustainability [1-3]. Symbiotic arbuscular mycorrhizal (AM) fungi are major players in helping plants growing under nutrient starvation conditions. They provide plants with minerals like phosphate and, furthermore, act as modulators of plant growth altering the root developmental program [4, 5]. However, the precise mechanisms involved in this latter process are not well understood. Here, we show that AM fungi are able to modulate root cortex development in Medicago truncatula by activating a novel GRAS-domain transcription factor, MIG1, that determines the size of cortical root cells. MIG1 expression peaks in arbuscule-containing cells, suggesting a role in cell remodeling during fungal accommodation. Roots ectopically expressing MIG1 become thicker due to an increase in the number and width of cortical cells. This phenotype is fully counteracted by gibberellin (GA) and phenocopied with a GA biosynthesis inhibitor or by expression of a dominant DELLA (Δ18DELLA1) protein. MIG1 downregulation leads to malformed arbuscules, a phenotype rescued by Δ18DELLA1, suggesting that MIG1 intersects with the GA signaling to control cell morphogenesis through DELLA1. DELLA1 was shown to be a central node controlling arbuscule branching [6-8]. Now we provide evidence that, together with MIG1, DELLA1 is responsible for radial cortical cell expansion during arbuscule development. Our data point toward DELLA proteins being not only longitudinal root growth repressors [9] but also positive regulators of cortical radial cell expansion, extending the knowledge of how DELLAs control root growth.

A tandem Kunitz protease inhibitor (KPI106)-serine carboxypeptidase (SCP1) controls mycorrhiza establishment and arbuscule development in Medicago truncatula.

Plant proteases and protease inhibitors are involved in plant developmental processes including those involving interactions with microbes. Here we show that a tandem between a Kunitz protease inhibitor (KPI106) and a serine carboxypeptidase (SCP1) controls arbuscular mycorrhiza development in the root cortex of Medicago truncatula. Both proteins are only induced during mycorrhiza formation and belong to large families whose members are also mycorrhiza-specific. Furthermore, the interaction between KPI106 and SCP1 analysed using the yeast two-hybrid system is specific, indicating that each family member might have a defined counterpart. In silico docking analysis predicted a putative P1 residue in KPI106 (Lys173) that fits into the catalytic pocket of SCP1, suggesting that KPI106 might inhibit the enzyme activity by mimicking the protease substrate. In vitro mutagenesis of the Lys173 showed that this residue is important in determining the strength and specificity of the interaction. The RNA interference (RNAi) inactivation of the serine carboxypeptidase SCP1 produces aberrant mycorrhizal development with an increased number of septated hyphae and degenerate arbuscules, a phenotype also observed when overexpressing KPI106. Protease and inhibitor are both secreted as observed when expressed in Nicotiana benthamiana epidermal cells. Taken together we envisage a model in which the protease SCP1 is secreted in the apoplast where it produces a peptide signal critical for proper fungal development within the root. KPI106 also at the apoplast would modulate the spatial and/or temporal activity of SCP1 by competing with the protease substrate.