Characterization of the molecular function of a novel effector of Sporisorium reilianum that impacts plant development

Agrawal, Nisha; Schirawski, Jan (Thesis advisor); van Dongen, Joost Thomas (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


Sporisorium reilianum is a biotrophic plant pathogen that causes head smut disease of maize. Symptoms include stunted growth, the formation of spores in the tassel and the cob, phyllody in the male and female organs, and formation of subapical ears caused by suppression of apical dominance of the maize ear. The effector protein SAD1 has been found to be responsible for suppressing apical dominance in maize without interfering with the auxin pathway. It interacts with ZmRGLG1 in the yeast two-hybrid library screening and causes the upregulation of abiotic stress-responsive genes in maize. To better understand how SAD1 impacts plant development, this thesis aimed to characterize the molecular interaction of SAD1 with the E3 ubiquitin ligases RGLG1 and RGLG2. With the help of a yeast secretion trap assay, I proved that SAD1 has a functional signal peptide, which suggests that SAD1 is secreted from S. reilianum into the plant. I successfully generated tagged versions of SAD1 expressing internal HA and hexahistidine tags that were able to complement the phenotype of ΔSAD1. These constructs can be used to study the in-planta localization of SAD1 in maize. Targeted yeast two-hybrid analyses showed that SAD1 interacts with ZmRGLG1 and ZmRGLG2, and their A. thaliana homologs AtRGLG1 and AtRGLG2. Bimolecular fluorescence complementation analysis showed that SAD1 interacts with all four E3 ubiquitin ligases in plant cells in the nucleus and the cytoplasm. The E3 ubiquitin ligases AtRGLG1 and AtRGLG2 are known to have a role in abiotic stress response. rglg1rglg2 double mutants of A. thaliana show loss of apical dominance. To study the role of the interaction between SAD1 and AtRGLG1/2, mCHERRY-SAD1 lacking its signal peptide was stably expressed under the control of the strong constitutive cauliflower mosaic virus 35S promoter (P35S:mcherry-SAD1ΔSP) in rglg1, rglg2, and Col-0 A. thaliana lines. The resulting transgenic lines displayed more secondary cauline-leaf branches and primary and secondary rosette-leaf branches. In contrast, root growth was unaffected in plate-germinated seedlings of any of the transformed lines. An in-vitro ubiquitination assay showed that SAD1 can be ubiquitinylated by RGLG1 and RGLG2 of both A. thaliana and Zea mays in the presence of the E2 ubiquitin-conjugating enzymes UbcH5b and UbcH13. I propose that SAD1 may interfere with the interaction of RGLG1 and RGLG2 with their natural targets, such as ERF53, that is typically targeted for degradation to turn off the prolonged abiotic stress response. This would lead to increased persistence of the stress-responsive transcription factor ERF53, which in turn would lead to prolonged-expression of abiotic stress response genes and loss of apical dominance. In addition to allocating a function to the fungal effector protein SAD1, these findings offer a new understanding of apical dominance in plants. My research results point to a conserved pathway regulating apical dominance and acting in both monocot and dicot plants independent of auxin that can be affected by SAD1. A better understanding of how apical dominance in inflorescences is achieved and how it can be modulated can also lead to an advancement in applied plant science, e.g., by developing crops that carry more ears, or ornamental plants that carry more flowers.