Intrinsically Disordered Kiwellin Protein-Like Effectors Target Plant Chloroplasts and are Extensively Present in Rust Fungi.

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.

The TATA-box sequence in the basal promoter contributes to determining light-dependent gene expression in plants.

A prototype 13-bp TATA-box sequence, TCACTATATATAG, was mutated at each nucleotide position and examined for its function in the core promoter. Specific nucleotides in the first TATA, the second TATA, as well as the flanking sequences influenced promoter function in transient transformation of tobacco (Nicotiana tabacum var Petit Havana) leaves. The effect of a given mutation on reporter gene expression in light versus dark was variable and sometimes contrasting. Some mutations, like T(7) or A(8)-->C or G, completely inactivated the expression of the minimal promoter in light but not in dark. In general, the sequence requirement for dark expression was less stringent than that for light expression. The selective effect of TATA-box mutations on light versus dark expression was exerted on core promoter function in the chromatin-integrated state also. Even in the presence of an upstream light response activator element, TATA-box mutations influenced modulation of the promoter by light. An A at the eighth position was specifically involved in the red light response of the promoter. Selectivity in gene expression was associated with a high level of transcript initiation from a site that was not active in the dark. Nuclear proteins from dark- and light-grown seedlings showed that the sequence variation within the TATA-box governs the formation of alternative transcriptional complexes. The experiments give direct evidence for the role of a core TATA-box sequence in determining the level as well as selectivity of gene expression in plants.

Analysis of polarity in the expression from a multifactorial bidirectional promoter designed for high-level expression of transgenes in plants.

A synthetic bidirectional expression module was constructed by placing a computationally designed minimal promoter sequence on the 5' and 3' sides of a transcription activation module. The activation of transcription from the unidirectional and bidirectional promoters constructed from the same sequence elements was evaluated by using the reporter genes gusA and gfp. The analysis based on transient and stable transformation of tobacco showed that the artificially designed multifactorial activation module activated transcription simultaneously to comparable levels in both the directions. The transcription activation module responded to elicitors like salicylic acid, NaCl and IAA in the forward as well as reverse directions. The concentration of the elicitor required for highest gene activation was similar for the two directions in case of the three activators. The kinetics of time of induction was similar in the two directions for salicylic acid and NaCl. In the case of IAA, the transcription activation was faster in the reverse direction. The results show that constitutive and chemically inducible bidirectional promoters can be deployed for predictable simultaneous regulation of two genes for genetic engineering in plants.

A variety of synergistic and antagonistic interactions mediated by cis-acting DNA motifs regulate gene expression in plant cells and modulate stability of the transcription complex formed on a basal promoter.

Several synthetic promoters containing a variety of commonly found cis-acting DNA sequence motifs were constructed to study the motif-motif and motif-protein interactions involved in gene expression in plants. Transient expression of the reporter gene gusA in tobacco leaves was used to demonstrate that several sequence elements can be arranged upstream of a basal promoter to function synergistically in enhancing gene expression. A cis-acting DNA motif could function as an activator by itself as well as a synergizing activator in the presence of other homologous as well as heterologous motifs in the neighbourhood. The function of a complex promoter comprising several activation motifs was arrested nearly completely in vivo, following titration with any one of the motifs. The results suggested a hierarchical assembly of several motif-binding factors, leading to the stabilization of the transcriptional complex formed on the TATA-box.