A dual regulatory role of the arbuscular mycorrhizal master regulator RAM1 in tomato.

The REQUIRED FOR ARBUSCULAR MYCORRHIZATION1 (RAM1) transcription factor from the GRAS family is well-known by its role as a master regulator of the arbuscular mycorrhizal (AM) symbiosis in dicot and monocot species, being essential in the transcriptional reprograming for the development and functionality of the arbuscules. In tomato, SlGRAS27 is the putative ortholog of RAM1 (here named SlRAM1), but has not yet been characterized. A reduced colonization of the root and an impaired arbuscule formation were observed in the SlRAM1 silenced plants, confirming the functional conservation of the RAM1 ortholog in tomato . However, unexpectedly, SlRAM1 overexpressing (UBIL:SlRAM1) plants also showed a decreased mycorrhizal colonization. Analysis of non-mycorrhizal UBIL:SlRAM1 roots revealed an overall regulation of AM-related genes and a reduction of strigolactone biosynthesis. Moreover, the external application of the strigolactone analogue GR244DO almost completely reversed the negative effects of SlRAM1 overexpression on the frequency of mycorrhization. However, it only partially recovered the pattern of arbuscule distribution observed in control plants. Our results strongly suggest that SlRAM1 has a dual regulatory role during mycorrhization and, apart from its recognized action as a positive regulator of arbuscule development, SlRAM1 is also involved in different mechanisms for the negative regulation of mycorrhization, including the repression of strigolactone biosynthesis.

The Role of CLE Peptides in the Suppression of Mycorrhizal Colonization of Tomato.

Symbioses with beneficial microbes are widespread in plants, but these relationships must balance the energy invested by the plants with the nutrients acquired. Symbiosis with arbuscular mycorrhizal (AM) fungi occurs throughout land plants, but our understanding of the genes and signals that regulate colonization levels is limited, especially in non-legumes. Here, we demonstrate that in tomato, two CLV3/EMBRYO-SURROUNDING REGION (CLE) peptides, SlCLE10 and SlCLE11, act to suppress AM colonization of roots. Mutant studies and overexpression via hairy transformation indicate that SlCLE11 acts locally in the root to limit AM colonization. Indeed, SlCLE11 expression is strongly induced in AM-colonized roots, but SlCLE11 is not required for phosphate suppression of AM colonization. SlCLE11 requires the FIN gene that encodes an enzyme required for CLE peptide arabinosylation to suppress mycorrhizal colonization. However, SlCLE11 suppression of AM does not require two CLE receptors with roles in regulating AM colonization, SlFAB (CLAVATA1 ortholog) or SlCLV2. Indeed, multiple parallel pathways appear to suppress mycorrhizal colonization in tomato, as double mutant studies indicate that SlCLV2 and FIN have an additive influence on mycorrhizal colonization. SlCLE10 appears to play a more minor or redundant role, as cle10 mutants did not influence intraradical AM colonization. However, the fact that cle10 mutants had an elevated number of hyphopodia and that ectopic overexpression of SlCLE10 did suppress mycorrhizal colonization suggests that SlCLE10 may also play a role in suppressing AM colonization. Our findings show that CLE peptides regulate AM colonization in tomato and at least SlCLE11 likely requires arabinosylation for activity.

Microtubule cytoskeleton and mycorrhizal roots.

For the establishment of the Arbuscular Mycorrhiza (AM) symbiosis it is essential that epidermis and cortical cells from plant roots suffer a strong reorganization to allow the penetration of intracellular fungal hyphae. In the same manner, the new formation of a periarbuscular membrane and a symbiotic interface with specific compositions are required for a functional symbiosis. It is believed that the cytoskeleton of the plant host plays an essential role in these processes, particularly the microtubule (MT) cytoskeleton, as huge modifications have been observed in the MT array of root cells accompanying the establishment of the AM symbiosis. Recent research has established a link between microtubule rearrangements and arbuscule functioning. However, further research is required to elucidate the specific functions of MT cytoskeleton along the different stages of the arbuscule life cycle and to unravel the signals triggering these changes.

DLK2 regulates arbuscule hyphal branching during arbuscular mycorrhizal symbiosis.

D14 and KAI2 receptors enable plants to distinguish between strigolactones (SLs) and karrikins (KARs), respectively, in order to trigger appropriate environmental and developmental responses. Both receptors are related to the regulation of arbuscular mycorrhiza (AM) formation and are members of the RsbQ-like family of α,ß-hydrolases. DLK2 proteins, whose function remains unknown, constitute a third clade from the RsbQ-like protein family. We investigated whether the tomato SlDLK2 is a new regulatory component in the AM symbiosis. Genetic approaches were conducted to analyze SlDLK2 expression and to understand SlDLK2 function in AM symbiosis. We show that SlDLK2 expression in roots is AM-dependent and is associated with cells containing arbuscules. SlDLK2 ectopic expression arrests arbuscule branching and downregulates AM-responsive genes, even in the absence of symbiosis; while the opposite effect was observed upon SlDLK2 silencing. Moreover, SlDLK2 overexpression in Medicago truncatula roots showed the same altered phenotype observed in tomato roots. Interestingly, SlDLK2 interacts with DELLA, a protein that regulates arbuscule formation/degradation in AM roots. We propose that SlDLK2 is a new component of the complex plant-mediated mechanism regulating the life cycle of arbuscules in AM symbiosis.

Histochemical Staining and Quantification of Arbuscular Mycorrhizal Fungal Colonization.

Histochemical staining and light microscopy-based techniques have been widely used to detect and quantify arbuscular mycorrhizal fungi (AMF) in roots. Here we describe a standardized method for staining of AMF in colonized roots, and we provide possible modifications to adjust the protocol according to particular requirements, such as the type of root material or the reduction of toxic products. In addition, we also summarize some of the most common ways to quantify arbuscular mycorrhizal colonization.