Partitioning the structural features that underlie expansin-like and elicitor activities of cerato-platanin.

Cerato-platanin family (CPF) proteins are produced by fungi and elicit defences when applied to plants, behaving as PAMPs/MAMPs. CPF proteins share structural similarity to plant and bacterial expansins, and have been demonstrated, in some cases, to possess expansin-like loosening activity on cellulose. This is the case of cerato-platanin (CP), the founder of the CPF, which shows both eliciting and cellulose-loosening activities, raising the question as to whether the expansin-like activity may be responsible for defence activation. To pinpoint structural and thermodynamic features underlying eliciting and expansin-like activity of CP, we carried out site-directed mutagenesis targeting separately net charge (N84D mutation), conformational stability (V63A mutation), or conserved position previously shown to affect expansin-like activity in CP (D77A mutation), and characterized wild-type protein and its variants. Removing or adding negative charges on the protein surface led to reducing or increasing, respectively, the expansin-like activity. The activity was instead not affected by mutations affecting protein fold and stability. In contrast, all the mutants showed reduced capacity to elicit defences in plants. We conclude that the expansin-like activity of CP depends on net charge and ability to (weakly) bind cellulose, whereas the eliciting activity on plants does not depend on the cellulose-loosening activity.

Accumulation patterns of endogenous ß-aminobutyric acid during plant development and defence in Arabidopsis thaliana.

We recently discovered that ß-aminobutyric acid (BABA), a molecule known for its ability to prime defences in plants, is a natural plant metabolite. However, the role played by endogenous BABA in plants is currently unknown. In this study we investigated the systemic accumulation of BABA during pathogen infection, levels of BABA during plant growth and development and analysed mutants possibly involved in BABA transport or regulation. BABA was quantified by LC-MS using an improved method adapted from a previously published protocol. Systemic accumulation of BABA was determined by analysing non-infected leaves and roots after localised infections with Plectosphaerella cucumerina or Pseudomonas syringae pv. tomato (Pst) DC3000 avrRpt2. The levels of BABA were also quantified in different plant tissues and organs during normal plant growth, and in leaves during senescence. Mutants affecting amino acid transport (aap6, aap3, prot1 and gat1), γ-aminobutyric acid levels (pop2) and senescence/defence (cpr5-2) were analysed. BABA was found to accumulate only locally after bacterial or fungal infection, with no detectable increase in non-infected systemic plant parts. In leaves, BABA content increased during natural and induced senescence. Reproductive organs had the highest levels of BABA, and the mutant cpr5-2 produced constitutively high levels of BABA. Synthetic BABA is highly mobile in the receiving plant, whereas endogenous BABA appears to be produced and accumulated locally in a tissue-specific way. We discuss a possible role for BABA in age-related resistance and propose a comprehensive model for endogenous and synthetic BABA.

A novel approach for the identification of efficient combination therapies in primary human acute myeloid leukemia specimens.

Appropriate culture methods for the interrogation of primary leukemic samples were hitherto lacking and current assays for compound screening are not adapted for large-scale investigation of synergistic combinations. In this study, we report a novel approach that efficiently distills synthetic lethal interactions between small molecules active on primary human acute myeloid leukemia (AML) specimens. In single-dose experiments and under culture conditions preserving leukemia stem cell activity, our strategy considerably reduces the number of tests needed for the identification of promising compound combinations. Initially conducted with a selected library of 5000 small molecules and 20 primary AML specimens, it reveals 5 broad classes of sensitized therapeutic target pathways along with their synergistic patient-specific fingerprints. This novel method opens new avenues for the development of AML personalized therapeutics and may be generalized to other tumor types, for which in vitro cancer stem cell cultures have been developed.