Tonoplast Sucrose Trafficking Modulates Starch Utilization and Water Deficit Behavior in Poplar Leaves.

Leaf osmotic adjustment by the active accrual of compatible organic solutes (e.g. sucrose) contributes to drought tolerance throughout the plant kingdom. In Populus tremula x alba, PtaSUT4 encodes a tonoplast sucrose-proton symporter, whose downregulation by chronic mild drought or transgenic manipulation is known to increase leaf sucrose and turgor. While this may constitute a single drought tolerance mechanism, we now report that other adjustments which can occur during a worsening water deficit are damped when PtaSUT4 is constitutively downregulated. Specifically, we report that starch use and leaf relative water content (RWC) dynamics were compromised when plants with constitutively downregulated PtaSUT4 were subjected to a water deficit. Leaf RWC decreased more in wild-type and vector control lines than in transgenic PtaSUT4-RNAi (RNA-interference) or CRISPR (clustered regularly interspersed short palindromic repeats) knockout (KO) lines. The control line RWC decrease was accompanied by increased PtaSUT4 transcript levels and a mobilization of sucrose from the mesophyll-enriched leaf lamina into the midvein. The findings suggest that changes in SUT4 expression can increase turgor or decrease RWC as different tolerance mechanisms to reduced water availability. Evidence is presented that PtaSUT4-mediated sucrose partitioning between the vacuole and the cytosol is important not only for overall sucrose abundance and turgor, but also for reactive oxygen species (ROS) and antioxidant dynamics. Interestingly, the reduced capacity for accelerated starch breakdown under worsening water-deficit conditions was correlated with reduced ROS in the RNAi and KO lines. A role for PtaSUT4 in the orchestration of ROS, antioxidant, starch utilization and RWC dynamics during water stress and its importance in trees especially, with their high hydraulic resistances, is considered.

Ustilago maydis phosphodiesterases play a role in the dimorphic switch and in pathogenicity.

Components of the cAMP (cyclic AMP) signalling cascades are conserved from fungi to humans, and are particularly important for fungal dimorphism and pathogenicity. Previous work has described two phosphodiesterases, UmPde1 and UmPde2, in Ustilago maydis which show strong phosphodiesterase activity. We further characterized the biological function(s) of these phosphodiesterases in U. maydis. Specifically, we examined their possible role(s) in regulation of the cAMP-dependent protein kinase A (PKA) pathway and their roles in filamentous growth and pathogenicity. We found that UmPde1, which shares 35 % similarity with Cryptococcus neoformans Pde1, also displays functional homology with this enzyme. UmPde1 complements the capsule-formation defect of C. neoformans strains deleted for Pde1. In U. maydis, the cell morphology of the umpde1 deletion mutant resembled the multiple budding phenotypes seen with the ubc1 mutant, which lacks the regulatory subunit of PKA. Interestingly, on low-ammonium medium, umpde2 deletion strains showed a reduction in filamentation that was comparable to that of ubc1 deletion strains; however, umpde1 deletion strains showed normal filamentation on low-ammonium medium. Furthermore, both the ubc1 deletion strain in which the PKA pathway was constitutively active and the umpde1 deletion strains were significantly reduced in pathogenicity, while the umpde2 deletion strains showed a trend for reduced pathogenicity compared with wild-type strains. These data support a role for the phosphodiesterases UmPde1 and UmPde2 in regulating the U. maydis cAMP-dependent PKA pathway through modulation of cAMP levels, thus affecting dimorphic growth and pathogenicity.

Role of Hsl7 in morphology and pathogenicity and its interaction with other signaling components in the plant pathogen Ustilago maydis.

The phytopathogenic fungus Ustilago maydis undergoes a dimorphic transition in response to mating pheromone, host, and environmental cues. On a solid medium deficient in ammonium (SLAD [0.17% yeast nitrogen base without ammonium sulfate or amino acids, 2% dextrose, 50 µM ammonium sulfate]), U. maydis produces a filamentous colony morphology, while in liquid SLAD, the cells do not form filaments. The p21-activated protein kinases (PAKs) play a substantial role in regulating the dimorphic transition in fungi. The PAK-like Ste20 homologue Smu1 is required for a normal response to pheromone, via upregulation of pheromone expression, and virulence, and its disruption affects both processes. Our experiments suggest that Smu1 also regulates cell length and the filamentous response on solid SLAD medium. Yeast two-hybrid analysis suggested an Hsl7 homologue as a potential interacting partner of Smu1, and a unique open reading frame for such an arginine methyltransferase was detected in the U. maydis genome sequence. Hsl7 regulates cell length and the filamentous response to solid SLAD in a fashion opposite to that of Smu1, but neither overexpression nor disruption of hsl7 attenuates virulence. Simultaneous disruption of hsl7 and overexpression of smu1 lead to a hyperfilamentous response on solid SLAD. Moreover, only this double mutant strain forms filaments in liquid SLAD. The double mutant strain was also significantly reduced in virulence. A similar filamentous response in both solid and liquid SLAD was observed in strains lacking another PAK-like protein kinase involved in cytokinesis and polar growth, Cla4. Our data suggest that Hsl7 may regulate cell cycle progression, while both Smu1 and Cla4 appear to be involved in the filamentous response in U. maydis.