As F. oxysporum mainly grows intercellularly during early stages, we hypothesized that the pathogen may secrete pathogenicity proteins, known as effectors, in this intercellular space for determining general compatibility and to deal with host immunity. As a part of my MSCA fellowship, I mainly established the isolation of apoplastic fluid from infected tomato roots and used discovery proteomics to identify effectors.
These identified ´core apoplastic effectors´ encode for small, secreted, cysteine rich hypothetical proteins, which are conserved across the Fusarium species complex (Redkar et al., in prep). These core effectors termed (ERCs: Effector for Root Compatibility ERC1-3), are induced in planta upon colonization of host/non-host and contribute in virulence. I also performed a comprehensive RNA-Seq analysis during the early stages of colonization by F. oxysporum and found that a major proportion of the secretome primarily encodes for effectors that are conserved across the Fusarium specific complex. This suggests that Fusarium rely on a conserved effector repertoire for establishing compatibility on hosts and non-host and then host-specific determinants may primarily play a role in determining wilting which is host-specific.
We also investigated on the nature of the active intercellular root proteome during Fusarium colonization and how its activity triggers defense signaling. Infected apoplastic fluid was used for Activity Based Protein Profiling (ABPP), which indicated that Fusarium is able to suppress different classes of proteases. Based on these findings and our interests in the apoplastic immunity, I lead a research spotlight article in Trends in Plant Science (doi.org/10.1016/j.tplants.2019.06.009). The paper highlights work on effector from Ustilago maydis Pit2, that has a protease inhibitory domain, and which mimics the protease substrate. Additionally, I also co-authored a review on the current knowledge about Molecular Interactions of the smut fungi and their hosts in Annual Reviews (doi: 10.1146/annurev-phyto-082718- 100139). This has impacted ongoing research in the plant-pathogen interaction community.
Moreover, I have used Arabidopsis- Fusarium patho-system to understand the host derived signals that the fungus senses in reaching xylem. The root endodermis is a protective cell layer which separates cortical zone and vasculature. The space between endodermal cells becomes impregnated with a lignin-based hydrophobic polymer, termed the Casparian strip (CS), and acts as a selective barrier. Cell wall-associated membrane proteins, (CASPs) fuse to form a continuous band that is guided by the well-defined Schengen (SGN) pathway. I hypothesize that vascular-wilts likely interfere with different components of the SGN pathway to sense the vasculature. We used a collection of Arabidopsis mutants across the SGN pathway, that show varying degrees of pathogenicity phenotype with F. oxysporum Fo5176. Hence, The CS pathway seems to be involved in guiding Fusarium towards the vasculature. Results of my project were presented at research seminars at CEPLAS Fellows Conference at University of Dusseldorf, Germany and IISER, Pune, India.
Moving forward, I will continue in my host lab through the Juan de la Cierva Fellowship. I am working to functionally characterise the identified CORE effectors in Fusarium. I will also explore the interesting leads of the interference of Fusarium with the Casparian Strip pathway in sensing vasculature. Upon completion, I aim to publish two research papers detailing my findings.
In addition to the proposed work, I was also the corresponding author of two commentary articles: one which describes how root pathogen have evolved to target master transcriptional regulators of the Salicylic acid pathway (doi:10.1098/rstb.2015.0459) and another which provides an overview of how pathogens have evolved to use carrier proteins for virulence functions (doi: 10.1111/nph.14137).
