Lauriebeth Leonelli’s research while affiliated with University of California, Berkeley and other places

Pucciniamonoica is a spectacular plant parasitic rust fungus that triggers the formation of flower-like structures (pseudoflowers) in its Brassicaceae host plant Boecherastricta. Pseudoflowers mimic in shape, color, nectar and scent co-occurring and unrelated flowers such as buttercups. They act to attract insects thereby aiding spore dispersal and sexual reproduction of the rust fungus. Although much ecological research has been performed on P. monoica-induced pseudoflowers, this system has yet to be investigated at the molecular or genomic level. To date, the molecular alterations underlying the development of pseudoflowers and the genes involved have not been described. To address this, we performed gene expression profiling to reveal 256 plant biological processes that are significantly altered in pseudoflowers. Among these biological processes, plant genes involved in cell fate specification, regulation of transcription, reproduction, floral organ development, anthocyanin (major floral pigments) and terpenoid biosynthesis (major floral volatile compounds) were down-regulated in pseudoflowers. In contrast, plant genes involved in shoot, cotyledon and leaf development, carbohydrate transport, wax biosynthesis, cutin transport and L-phenylalanine metabolism (pathway that results in phenylethanol and phenylacetaldehyde volatile production) were up-regulated. These findings point to an extensive reprogramming of host genes by the rust pathogen to induce floral mimicry. We also highlight 31 differentially regulated plant genes that are enriched in the biological processes mentioned above, and are potentially involved in the formation of pseudoflowers. This work illustrates the complex perturbations induced by rust pathogens in their host plants, and provides a starting point for understanding the molecular mechanisms of pathogen-induced floral mimicry.

Clustal alignment of 16 isolates of ATR13 showing amino acid percent conservation. Arrows denote helices, blue line indicates coiled regions, and the red line denotes disordered residues. (TIF)

Alternative low-energy structures selected from the family of 20 NMR structures of ATR13 Emco5. A–D. Various representations of the ATR13 structure and their 180° rotations. Polymorphic residues are shown in orange and conserved residues are depicted in blue. (TIF)

Various amino acid changes in ATR13 Emco5 that disrupt RPP13 recognition. Wildtype residue identities are listed in bold next to their amino acid position. LOR mutants generated by PCR random mutatgenesis are listed in colors corresponding to whether those changes occurred as single (red), double (orange), or triple mutations (blue). Retention of recognition (ROR) mutants are listed in black next to the loss of function mutants and possess intact RPP13Nd recognition, illustrating that amino acid position's tolerance for change. (TIFF)

Random loss-of-recognition (LOR) mutagenesis of ATR13 Emco5 scored for HR in RPP13Nd transgenic N. benthamiana plants. A. Inoculations of various mutants generated by random pcr mutagenesis showing the varied timing and intensity of HR response after 24 h and 72 h. B. Western blot of various clones probed with α-ATR13 and ponceau for loading. C. A key to inoculation and expression data, consolidating complete lack of HR (red font), wildtype protein expression (yellow boxes), and residue alterations. Mutant clones marked with an asterisk had unique mutations not present in the retention-of-function mutational database or in other clones. (TIF)

Crystals of ATR13. A. Examples of Δ53 ATR13 Emco5 crystals grown in various conditions in sitting drop trays. B. ATR13 crystal grown in chip format (Fluidigm, Inc). C. Typical diffraction pattern associated with ATR13 crystals. (TIF)

Overlay of ATR13 structure and Ran-GTP. A. ATR13 is shown in magenta and RAN-GTP is in blue, showing that the homology here is incidental and not significant. B. Rotated view of the overlay of ATR13 and RAN-GTP. (TIF)

Bacterial growth assays on Pu2-23, Est-1 and Ws-2. Additional bacterial growth assays showing recognition of different ATR1 alleles by Arabidopsis accessions (A) Pu2-23, (B) Est-1 and (C) Ws-2. (PDF)

Images of the trypan blue-stained Arabidopsis cotyledons and true leaves inoculated with Hpa Emco5. (PDF)

Phenotypic responses of 83 Arabidopsis accessions to H. arabidopsidis strains Emoy2, Maks9, Emco5, Cala2 and Emwa1. Accessions are listed in alphabetical order. Coloring scheme: brown – absence of asexual sporulation on both cotyledons and true leaves, orange – sporulation is present on cotyledons, but not on true leaves, yellow – sporulation is present on both cotyledons and true leaves. Numbers indicate phenotypic scoring (type 1 to 5, described in the text) for the interactions that have been analyzed by microscopy (see Datasets S1, S2, S3, S4, S5), n – data not available. The first number in each column corresponds to the score on cotyledons, the second number to the score on true leaves. (XLS)