RESUMO
The effector proteins produced by plant pathogens are one of the essential components of host-pathogen interaction. Despite being important, most of the effector proteins remain unexplored due to the diversity in their primary sequence generated by the high selection pressure of the host immune system. However to maintain the primary function in the infection process, these effectors may tend to maintain their native protein fold to perform the corresponding biological function. In the present study, unannotated candidate secretory effector proteins of sixteen major plant fungal pathogens were analyzed to find the conserved known protein folds using homology, ab initio, and Alpha Fold/Rosetta Fold protein dimensional (3D) structure approaches. Several unannotated candidate effector proteins were found to match various known conserved protein families potentially involved in host defense manipulation in different plant pathogens. Surprisingly a large number of plant Kiwellin proteins fold like secretory proteins (> 100) were found in studied rust fungal pathogens. Many of them were predicted as potential effector proteins. Furthermore, template independent modelling using Alpha Fold/Rosetta Fold analysis and structural comparison of these candidates also predicted them to match with plant Kiwellin proteins. We also found plant Kiwellin matching proteins outside rusts including several non-pathogenic fungi suggesting the broad function of these proteins. One of the highest confidently modeled Kiwellin matching candidates effectors, Pstr_13960 (97.8%), from the Indian P. striiformis race Yr9 was characterized using overexpression, localization, and deletion studies in Nicotiana benthamiana. The Pstr_13960 suppressed the BAX-induced cell death and localized in the chloroplast. Furthermore, the expression of the Kiwellin matching region (Pst_13960_kiwi) alone suppressed the BAX-induced cell death in N. benthamiana despite the change of location to the cytoplasm and nucleus, suggesting the novel function of the Kiwellin core fold in rust fungi. Molecular docking showed that Pstr_13960 can interact with plant Chorismate mutases (CMs) using three loops conserved in plant and rust Kiwellins. Further analysis of Pstr_13960 showed to contain Intrinsically disordered regions (IDRs) in place of the N-terminal ß1/ß2 region found in plant Kiwellins suggesting the evolution of rust Kiwellins-like effectors (KLEs). Overall, this study reports the presence of a Kiwellin protein-like fold containing a novel effector protein family in rust fungi depicting a classical example of the evolution of effectors at the structure level as Kiwellin effectors show very low significant similarity to plant Kiwellin at the sequence level.
RESUMO
Deepwater is an abiotic stress that limits rice cultivation worldwide due to recurrent floods. The miRNAs and lncRNAs are two non-coding RNAs emerging as major regulators of gene expressions under different abiotic stresses. However, the regulation of these two non-coding RNAs under deepwater stress in rice is still unexplored. In this study, small RNA-seq and RNA-seq from internode and node tissues were analyzed to predict deepwater stress responsive miRNAs and lncRNAs, respectively. Additionally, a competitive endogenous RNA (ceRNA) study revealed about 69 and 25 lncRNAs acting as endogenous target mimics (eTM) with the internode and node miRNAs, respectively. In ceRNA analyses, some of the key miRNAs such as miR1850.1, miR1848, and IN-nov-miR145 were upregulated while miR159e was downregulated, and their respective eTM lncRNAs and targets were found to have opposite expressions. Moreover, we have transiently expressed one module (IN-nov-miR145-Cc-TCONS_00011544-Os11g36430.3) in tobacco leaves. The integrated analysis has identified differentially expressed (DE) miRNAs, lncRNAs and their target genes, and the complex regulatory network, which might lead to stem elongation under deepwater stress. In this novel attempt to identify and characterize miRNAs and lncRNAs under deepwater stress in rice, we have provided, probably for the first time, a reference platform to study the interactions of these two non-coding RNAs with respective target genes through transient expression analyses.