Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.07.14.664668

Molecular identification and functional analysis of HrpZ2, a new member of harpin superfamily from Pseudomonas syringae inducing hypersensitive response in tobacco.

Lal, K.·Joshi, A.·Saini, V.·Mohammed, M.·Sarma, P.·Dey, D.
View original source ↗Export metadata ↓

Abstract

Harpins belong to a group of proteins with distinctive features such as heat stability, glycine richness, and absence of cysteine, and are secreted by many Gram-negative phytopathogens via the type III secretion system. Harpins are known to trigger hypersensitive response followed by induction of systemic acquired resistance in non-host plants. However, the molecular mechanism of harpin-induced hypersensitive response remained largely unexplored, mainly because of lack of structural information. In this study, we report the cloning of a new harpin gene (hrpZ2) from Pseudomonas syringae strain MTCC-11950, belonging to the harpin superfamily. In silico analysis revealed that about 50.29% of the protein consists of alpha-helices, 48.53% are random coils, and only 1.16% are beta-sheets, and nearly half (42%) of the protein consists of intrinsically disordered regions. Based on a prokaryotic predictive model and the presence of a signal peptide on its N-terminus, the subcellular localization of harpin is predicted as extracellular. To date, no experimentally determined crystal structure of any harpin protein is available. Therefore, we built and validated a three-dimentional model (with 99% of residues in allowed/additionally allowed regions and a Z-score of -5.3) of harpin. Phylogenetic analysis and functional domain studies revealed that this new harpin belongs to the harpin superfamily. Infiltration of harpin in tobacco leaves resulted in hypersensitive response, which was associated with oxidative burst, callose deposition, localized cell death, and increased activity of defense-related enzymes such as phenylalanine ammonia-lyase and polyphenol oxidase. Furthermore, infiltration of harpin in non-host plants from different angiosperm families induced hypersensitive response, indicating broad-spectrum agricultural applicability of this new harpin protein. This study elucidates the molecular and functional properties of the new harpin protein and its ability to induce hypersensitive response across a broad range of non-host plants.

Source connections

Explore related subjects

plant biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Lal, K., Joshi, A., Saini, V., Mohammed, M., Sarma, P., Dey, D.. 2025-07-14. Molecular identification and functional analysis of HrpZ2, a new member of harpin superfamily from Pseudomonas syringae inducing hypersensitive response in tobacco.. https://doi.org/10.1101/2025.07.14.664668

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Loss of starch synthase IIa alleviates the negative impact of high temperature on rice starch during grain filling

High temperatures during grain filling stage are becoming increasingly frequent, compromising both grain and eating quality and thereby driving demand for heat-resilient cultivars. Such conditions are known to reduce the expression of granule-bound starch synthase I (GBSSI) and starch branching enzyme IIb (BEIIb), which are involved in starch biosynthesis, resulting in a decrease in amylose content and an increase in long-chain amylopectin. Thus, the present study introduced functional mutation in starch synthase IIa (SSIIa) that increases the proportion of short amylopectin chains to genetically compensate for the high-temperature-induced increase in amylopectin long chain. Rice lines carrying the ss2a mutation were grown at two locations with cooler (Akita) and warmer (Okayama) temperatures. Their grain traits, starch structure, and eating quality were compared. The ss2a mutant lines showed an increased proportion of short amylopectin chains as well as an increased apparent amylose content. Furthermore, these alterations in starch structure varied with the grain-filling temperature of the cultivation sites, ultimately affected eating quality. These results suggest that enriching short amylopectin chain via the ss2a mutation can counteract the increase in long amylopectin chain caused by high temperatures during grain filling, thereby maintaining a desirable starch structure and eating quality.

plant biology↗
bioRxiv2026

Analysis of SpCas9 on- and off-target effects in high efficiency multiplex editing in Arabidopsis

RNA-guided nucleases (RGNs), such as Cas9 from Streptococcus pyogenes (SpCas9), are widely used for plant genome editing. Previous surveys for off-targeting, the modification of unintended targets with similarity to the intended target, indicate high specificity of SpCas9 in plant cells. However, off-targeting has not been assessed for efficiency-optimized editing systems combined with extensive multiplexing, which might increase the likelihood of cleavage at unintended sites. We therefore analyzed Arabidopsis thaliana lines that had been extensively mutagenized using zCas9i and up to 29 gRNAs addressing >45 target sites over several rounds of editing. Genomes were sequenced by short- and long-read technologies, and genome-wide variants were catalogued. Our pipeline for variant calling reliably detected RGN-induced mutations at on-targets. When excluding these on-target modifications, variants were detected in edited lines at frequencies similar to those previously reported for spontaneous mutations. In further analyses, we did not find any evidence for an origin of these variants from RGN activity. Our data are thus consistent with high specificity of SpCas9. In contrast, we detected genomic reorganization events upon editing at two complex loci, RPP1 and RPP7, encompassing multiple homologous genes, and also identified an allele by WGS that had escaped detection by amplicon sequencing. We conclude that, while off-targets may efficiently be avoided by selection of specific gRNAs, on-target modifications may be more extensive than intended, especially at complex loci and/or during multiplexing.

plant biology↗
bioRxiv2026

A cellulose synthase interactome uncovers BAG proteins as regulators of cellulose synthase homeostasis

Cellulose synthase complexes build the load-bearing cellulose microfibrils of plant cell walls, yet how the abundance of their catalytic CELLULOSE SYNTHASE A (CESA) subunits is maintained remains unclear. Here, we used multi-bait TurboID proximity labelling with ten cellulose-synthesis-associated baits and six subcellular controls to define a high-confidence cellulose synthase neighbourhood. Stringent spatial and recurrence-based filtering yielded a core network of 119 interactions among 44 proteins and identified three members of the conserved Bcl-2-associated athanogene (BAG) family as previously unrecognised regulators of cellulose synthase homeostasis. BAG1-3 associated with primary-wall CESAs in reciprocal proximity-labelling experiments. Arabidopsis bag mutants showed reduced cellulose accumulation, hypersensitivity to cellulose-synthesis inhibitors, and markedly decreased CESA protein abundance without corresponding changes in CESA transcript levels. Loss of BAG function also increased the accumulation of CESA6 in vacuolar compartments. These findings identify BAG proteins as previously unrecognised regulators of cellulose synthase homeostasis and link a conserved proteostasis-associated protein family to plant cell wall biosynthesis. More broadly, the study establishes multi-bait proximity labelling, combined with cell location-specific controls, as a strategy for resolving dynamic protein networks whose components traffic through multiple subcellular compartments.

plant biology↗