Prithwi Ghosh

Legumes can establish a mutual association with soil-derived nitrogen-fixing bacteria called ‘rhizobia’ forming lateral root organs called root nodules. Rhizobia inside the root nodules get transformed into ‘bacteroids’ that can fix atmospheric nitrogen to ammonia for host plants in return for nutrients and shelter. A substantial 200 million tons of nitrogen is fixed annually through biological nitrogen fixation. Consequently, the symbiotic mechanism of nitrogen fixation is utilized worldwide for sustainable agriculture and plays a crucial role in the Earth’s ecosystem. The development of effective nitrogen-fixing symbiosis between legumes and rhizobia is very specialized and requires coordinated signaling. A plethora of plant-derived nodule-specific cysteine-rich (NCR or NCR-like) peptides get actively involved in this complex and tightly regulated signaling process of symbiosis between some legumes of the IRLC (Inverted Repeat-Lacking Clade) and Dalbergioid clades and nitrogen-fixing rhizobia. Recent progress has been made in identifying two such peptidases that actively prevent bacterial differentiation, leading to symbiotic incompatibility. In this review, we outlined the functions of NCRs and two nitrogen-fixing blocking peptidases: HrrP (host range restriction peptidase) and SapA (symbiosis-associated peptidase A). SapA was identified through an overexpression screen from the Sinorhizobium meliloti 1021 core genome, whereas HrrP is inherited extra-chromosomally. Interestingly, both peptidases affect the symbiotic outcome by degrading the NCR peptides generated from the host plants. These NCR-degrading peptidases can shed light on symbiotic incompatibility, helping to elucidate the reasons behind the inefficiency of nitrogen fixation observed in certain groups of rhizobia with specific legumes.

Context Protein–protein interaction (PPI) is a key component linked to virtually all cellular processes. Be it an enzyme catalysis (‘classic type functions’ of proteins) or a signal transduction (‘non-classic’), proteins generally function involving stable or quasi-stable multi-protein associations. The physical basis for such associations is inherent in the combined effect of shape and electrostatic complementarities (Sc, EC) of the interacting protein partners at their interface, which provides indirect probabilistic estimates of the stability and affinity of the interaction. While Sc is a necessary criterion for inter-protein associations, EC can be favorable as well as disfavored (e.g., in transient interactions). Estimating equilibrium thermodynamic parameters (∆G_binding, K_d) by experimental means is costly and time consuming, thereby opening windows for computational structural interventions. Attempts to empirically probe ∆G_binding from coarse-grain structural descriptors (primarily, surface area based terms) have lately been overtaken by physics-based, knowledge-based and their hybrid approaches (MM/PBSA, FoldX, etc.) that directly compute ∆G_binding without involving intermediate structural descriptors. Methods Here, we present EnCPdock (https://www.scinetmol.in/EnCPdock/), a user-friendly web-interface for the direct conjoint comparative analyses of complementarity and binding energetics in proteins. EnCPdock returns an AI-predicted ∆G_binding computed by combining complementarity (Sc, EC) and other high-level structural descriptors (input feature vectors), and renders a prediction accuracy comparable to the state-of-the-art. EnCPdock further locates a PPI complex in terms of its {Sc, EC} values (taken as an ordered pair) in the two-dimensional complementarity plot (CP). In addition, it also generates mobile molecular graphics of the interfacial atomic contact network for further analyses. EnCPdock also furnishes individual feature trends along with the relative probability estimates (Pr_fmax) of the obtained feature-scores with respect to the events of their highest observed frequencies. Together, these functionalities are of real practical use for structural tinkering and intervention as might be relevant in the design of targeted protein-interfaces. Combining all its features and applications, EnCPdock presents a unique online tool that should be beneficial to structural biologists and researchers across related fraternities.

Context: Protein-protein interaction (PPI) is a key component linked to virtually all cellular processes. Be it an enzyme catalysis (‘ classic type functions ’ of proteins) or a signal transduction (‘ non-classic’ ), proteins generally function involving stable or quasi-stable multi-protein associations. The physical basis for such associations is inherent in the combined effect of shape and electrostatic complementarities (Sc, EC) of the interacting protein partners at their interface, which provides indirect probabilistic estimates of the stability and affinity of the interaction. While Sc is a necessary criterion for inter-protein associations, EC can be favorable as well as disfavored (e.g., in transient interactions). Estimating equilibrium thermodynamic parameters (ΔG _binding , K _d ) by experimental means is costly and time consuming, thereby opening windows for computational structural interventions. Attempts to empirically probe ΔG _binding from coarse-grain structural descriptors (primarily, surface area based terms) have lately been overtaken by physics-based, knowledge-based and their hybrid approaches (MM/PBSA, FoldX etc.) that directly compute ΔG _binding without involving intermediate structural descriptors. Methods: Here we present EnCPdock ( www.scinetmol.in/EnCPdock/ ), a user-friendly web-interface for the direct conjoint comparative analyses of complementarity and binding energetics in proteins. EnCPdock returns an AI-predicted ΔG _binding computed by combining complementarity (Sc, EC) and other high-level structural descriptors (input feature vectors), and, renders a prediction accuracy comparable to the state-of-the-art. EnCPdock further locates a PPI complex in terms of its {Sc, EC} values (taken as an ordered pair) in the two-dimensional Complementarity Plot (CP). In addition, it also generates mobile molecular graphics of the interfacial atomic contact network for further analyses. EnCPdock also furnishes individual feature trends along with the relative probability estimates (Pr _fmax ) of the obtained feature-scores with respect to the events of their highest observed frequencies. Together, these functionalities are of real practical use for structural tinkering and intervention as might be relevant in the design of targeted protein-interfaces. Combining all its features and applications, EnCPdock presents a unique online tool that should be beneficial to structural biologists and researchers across related fraternities.

The COVID-19 origin debate has greatly been influenced by genome comparison studies of late, revealing the emergence of the Furin-like cleavage site at the S1/S2 junction of the SARS-CoV-2 Spike (FLCS_Spike) containing its ₆₈₁PRRAR₆₈₅ motif, absent in other related respiratory viruses. Being the rate-limiting (i.e., the slowest) step, the host Furin cleavage is instrumental in the abrupt increase in transmissibility in COVID-19, compared to earlier onsets of respiratory viral diseases. In such a context, the current paper entraps a ‘disorder-to-order transition’ of the FLCS_Spike (concomitant to an entropy arrest) upon binding to Furin. The interaction clearly seems to be optimized for a more efficient proteolytic cleavage in SARS-CoV-2. The study further shows the formation of dynamically interchangeable and persistent networks of salt-bridges at the Spike–Furin interface in SARS-CoV-2 involving the three arginines (R682, R683, R685) of the FLCS_Spike with several anionic residues (E230, E236, D259, D264, D306) coming from Furin, strategically distributed around its catalytic triad. Multiplicity and structural degeneracy of plausible salt-bridge network archetypes seem to be the other key characteristic features of the Spike–Furin binding in SARS-CoV-2, allowing the system to breathe—a trademark of protein disorder transitions. Interestingly, with respect to the homologous interaction in SARS-CoV (2002/2003) taken as a baseline, the Spike–Furin binding events, generally, in the coronavirus lineage, seems to have preference for ionic bond formation, even with a lesser number of cationic residues at their potentially polybasic FLCS_Spike patches. The interaction energies are suggestive of characteristic metastabilities attributed to Spike–Furin interactions, generally to the coronavirus lineage, which appears to be favorable for proteolytic cleavages targeted at flexible protein loops. The current findings not only offer novel mechanistic insights into the coronavirus molecular pathology and evolution, but also add substantially to the existing theories of proteolytic cleavages.

The COVID-19 origin debate has greatly been influenced by Genome comparison studies of late, revealing the seemingly sudden emergence of the Furin-Like Cleavage Site at the S1/S2 junction of the SARS-CoV-2 Spike (FLCS _Spike ) containing its ₆₈₁ PRRAR ₆₈₅ motif, absent in other related respiratory viruses. Being the rate-limiting (i.e., the slowest) step, the host Furin cleavage is instrumental in the abrupt increase in transmissibility in COVID-19, compared to earlier onsets of respiratory viral diseases. In such a context, the current paper entraps a ’disorder-to-order transition’ of the FLCS _Spike (concomitant to an entropy arrest) upon binding to Furin. The interaction clearly seems to be optimized for a more efficient proteolytic cleavage in SARS-CoV-2. The study further shows the formation of dynamically interchangeable and persistent networks of salt-bridges at the Spike–Furin interface in SARS-CoV-2 involving the three arginines (R682, R683, R685) of the FLCS _Spike with several anionic residues (E230, E236, D259, D264, D306) coming from Furin, strategically distributed around its catalytic triad. Multiplicity and structural degeneracy of plausible salt-bridge network archetypes seems the other key characteristic features of the Spike–Furin binding in SARS-CoV-2 allowing the system to breathe – a trademark of protein disorder transitions. Interestingly, with respect to the homologous interaction in SARS-CoV (2002/2003) taken as a baseline, the Spike–Furin binding events generally in the coronavirus lineage seems to have a preference for ionic bond formation, even with lesser number of cationic residues at their potentially polybasic FLCS _Spike patches. The interaction energies are suggestive of a characteristic metastabilities attributed to Spike–Furin interactions generally to the coronavirus lineage – which appears to be favorable for proteolytic cleavages targeted at flexible protein loops. T he current findings not only offer novel mechanistic insights into the coronavirus molecular pathology and evolution but also add substantially to the existing theories of proteolytic cleavages.

Some soil bacteria called rhizobia can interact symbiotically with legumes in which they form nodules on the plant roots where they can reduce atmospheric dinitrogen to ammonia, a form of nitrogen that can be used by growing plants. Rhizobia/plant combinations can differ in how successful this symbiosis is—Sinorhizobium meliloti Rm1021 forms a relatively ineffective symbiosis with Medicago truncatula Jemalong A17 but Sinorhizobium medicae WSM419 is able to support more vigorous plant growth. Using proteomic data from free-living and symbiotic S. medicae WSM419, we previously identified a subset of proteins that were not closely related to any S. meliloti Rm1021 proteins and speculated that adding one or more of these proteins to S. meliloti Rm1021 would increase its effectiveness on M. truncatula A17. Three genes, Smed_3503, Smed_5985, and Smed_6456, were cloned into S. meliloti Rm1021 downstream of the E. colilacZ promoter. Strains with these genes increased nodulation and improved plant growth, individually and in combination with one another. Smed_3503, renamed iseA (increased symbiotic effectiveness) had the largest impact, increasing M. truncatula biomass by 61%. iseA homologs were present in all currently sequenced S. medicae strains but were infrequent in other Sinorhizobium isolates. Rhizobium leguminosarum bv. viciae 3841 containing iseA led to more nodules on pea and lentil. Split root experiments with M. truncatula A17 indicated that S. meliloti Rm1021 carrying the S. medicae iseA is less sensitive to plant induced resistance to rhizobial infection, suggesting an interaction with the plant’s regulation of nodule formation. IMPORTANCE The legume symbiosis with rhizobia is highly specific. Rhizobia that can nodulate and fix nitrogen on one legume species are often unable to associate with a different species. The interaction can be more subtle—symbiotically enhanced growth of the host plant can differ substantially when nodules are formed by different rhizobial isolates of a species, much like disease severity can differ when conspecific isolates of pathogenic bacteria infect different cultivars. Much is known about bacterial genes essential for a productive symbiosis, but less is understood about genes that marginally improve performance. We used a proteomic strategy to identify Sinorhizobium genes that contribute to plant growth differences that are seen when two different strains nodulate M. truncatula A17. These genes could also alter the symbiosis between R. leguminosarum bv. viciae 3841 and pea or lentil, suggesting that this approach may identify new genes that may more generally contribute to symbiotic productivity.

Two chiral Schiff base ligands 2-((1-hydroxy-3-phenylpropan-2-ylimino)methyl)-6-methoxyphenol (L¹H₂ ) and 2-(4-hydroxy-3-isopropylbut-1-enyl)-6-methoxyphenol (L²H₂ ) have been synthesized by the condensation of l-phenylalaninol/l-valinol and o-vanillin (2-hydroxy-3-methoxy benzaldehyde). A tetranuclear homometallic Cu(II) complex [Cu₄(L¹H)₂(L¹)₂] (ClO₄)₂ (C1) and a hexanuclear heterometallic complex [Cu₄(L²)₄Na₂(DMF)₂(H₂O)] (ClO₄)₂ (C2) have been synthesized with the ligands. Both the complexes possess cubane like Cu₄O₄ core with interesting structural variations and inherit the chirality of their corresponding ligands. The catalytic potential of the complexes has been explored for the oxidative kinetic resolution of racemic benzoin. The electronic, optical and chiroptical properties of the ligands and the complexes have been studied by DFT and TD-DFT calculations. Graphical Abstract Two chiral tetranuclear Cu(II) complexes have been synthesized from chiral Schiff base ligands. There are structural, spectroscopic and computational evidence of chirality of the complexes. The complexes show moderate activity toward enantioselective kinetic resolution of racemic benzoin with a maximum of 63% enantiomeric excess.

Main conclusion In this review, we have included evolution of plant intracellular immune receptors, oligomeric complex formation, enzymatic action, engineering, and mechanisms of immune inspection for appropriate defense outcomes. Abstract NLR (Nucleotide binding oligomerization domain containing leucine-rich repeat) proteins are the intracellular immune receptors that recognize pathogen-derived virulence factors to confer effector-triggered immunity (ETI). Activation of plant defense by the NLRs are often conveyed through N-terminal Toll-like/ IL-1 receptor (TIR) or non-TIR (coiled-coils or CC) domains. Homodimerization or self-association property of CC/ TIR domains of plant NLRs contribute to their auto-activity and induction of in planta ectopic cell death. High resolution crystal structures of Arabidopsis thaliana RPS4^TIR, L6^TIR, SNC1^TIR, RPP1^TIR and Muscadinia rotundifolia RPV1^TIR showed that interaction is mediated through one or two distinct interfaces i.e., αA and αE helices comprise AE interface and αD and αE helices were found to form DE interface. By contrast, conserved helical regions were determined for CC domains of plant NLRs. Evolutionary history of NLRs diversification has shown that paired forms were originated from NLR singletons. Plant TIRs executed NAD⁺ hydrolysis activity for cell death promotion. Plant NLRs were found to form large oligomeric complexes as observed in animal inflammasomes. We have also discussed different protein engineering methods includes domain shuffling, and decoy modification that increase effector recognition spectrum of plant NLRs. In summary, our review highlights structural basis of perception of the virulence factors by NLRs or NLR pairs to design novel classes of plant immune receptors.

Background: Suppression and activation of plant defense genes is comprehensively regulated by WRKY family transcription factors. Chickpea, the non-model crop legume suffers from wilt caused by Fusarium oxysporum f. sp. ciceri Race1 (Foc1), defense response mechanisms of which are poorly understood. Here, we attempted to show interaction between WRKY70 and several downstream signaling components involved in susceptibility/resistance response in chickpea upon challenge with Foc1. Results: In the present study, we found Cicer arietinum L. WRKY70 (CaWRKY70) negatively governs multiple defense responsive pathways, including Systemic Acquired Resistance (SAR) activation in chickpea upon Foc1 infection. CaWRKY70 is found to be significantly accumulated at shoot tissues of susceptible (JG62) chickpea under Foc1 stress and salicylic acid (SA) application. CaWRKY70 overexpression promotes susceptibility in resistant chickpea (WR315) plants to Foc1 infection. Transgenic plants upon Foc1 inoculation demonstrated suppression of not only endogenous SA concentrations but expression of genes involved in SA signaling. CaWRKY70 overexpressing chickpea roots exhibited higher ion-leakage and Foc1 biomass accumulation compared to control transgenic (VC) plants. CaWRKY70 overexpression suppresses H₂O₂ production and resultant reactive oxygen species (ROS) induced cell death in Foc1 infected chickpea roots, stem and leaves. Being the nuclear targeted protein, CaWRKY70 suppresses CaMPK9-CaWRKY40 signaling in chickpea through its direct and indirect negative regulatory activities. Protein-protein interaction study revealed CaWRKY70 and CaRPP2-like CC-NB-ARC-LRR protein suppresses hyper-immune signaling in chickpea. Together, our study provides novel insights into mechanisms of suppression of the multiple defense signaling components in chickpea by CaWRKY70 under Foc1 stress. Conclusion: CaWRKY70 mediated defense suppression unveils networking between several immune signaling events negatively affecting downstream resistance mechanisms in chickpea under Foc1 stress.