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Title: The knottin-like Blufensin family regulates genes involved in nuclear import and the secretory pathway in barley-powdery mildew interactions

item XU, WEIHUI - Iowa State University
item MENG, YAN - Iowa State University
item SURANA, PRIYANKA - Iowa State University
item Fuerst, Gregory
item NETTLETON, DAN - Iowa State University
item Wise, Roger

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/21/2015
Publication Date: 6/4/2015
Citation: Xu, W., Meng, Y., Surana, P., Fuerst, G.S., Nettleton, D., Wise, R.P. 2015. The knottin-like Blufensin family regulates genes involved in nuclear import and the secretory pathway in barley-powdery mildew interactions. Frontiers in Plant Science. 6:409. DOI:10.3389/fpls.2015.00409.

Interpretive Summary: Crop loss caused by disease remains one of the greatest challenges in agriculture in both developed and developing countries. Pathogenic fungi, viruses, bacteria, insects, and nematodes parasitize agronomic and horticultural crops, as well as commercial and recreational forests. Certain pathogens, like powdery mildews, require living host tissue to survive. Commonly, pathogens use small molecules, called effectors, to suppress, modify, or evade host defense. We recently characterized a new family of small cysteine-rich peptides from barley, designated blufensins (BLN), which interfere with defense against fungal pathogens. The genes that encode these peptides are unique to the cereal grain crops barley, wheat, and rice, and the resulting proteins are similar to knottins, a diverse family of proteins characterized by a unique "disulfide through disulfide knot". These results establish a previously unrecognized role for small peptides as negative regulators of plant defense, and as such, interactors or partners of BLN would be expected to play key roles in mediating the plant immune response. Therefore, to discern regulatory partners of BLN, we conducted genome-wide gene expression analysis of plants that had a Bln gene turned off by a technique called Virus Induced Gene Silencing; in this way one can determine what other proteins that may be influenced by the action of BLN1. Indeed, proteins implicated in nuclear import and the secretory pathway were found, two pathways critical to the genetic brigade when a plant defends itself against pathogen attack. This is significant because it indicates that there are still unknown interacting factors that connect resistance proteins and their downstream effects on the plant phenotype, thus, the functional identification of their precise roles will be a significant step in understanding plant defense. Because common themes govern all plant-pathogen interactions, this finding provides new knowledge of broad significance to plant scientists, and to growers who utilize disease resistance to protect their crops.

Technical Abstract: Plants have evolved complex regulatory mechanisms to control a multi-layered defense response to microbial attack. Both temporal and spatial gene expression are tightly regulated in response to pathogen ingress, modulating both positive and negative control of defense. BLUFENSINs, small knottin-like peptides in barley, wheat, and rice, are highly induced by attack from fungal pathogens, in particular, the obligate biotrophic fungus, Blumeria graminis f. sp. hordei (Bgh), causal agent of barley powdery mildew. Previous research indicated that Blufensin1 (Bln1) functions as a negative regulator of plant defense. In the current report, we show that BLN1 and BLN2 are both secreted into the apoplast and Barley stripe mosaic virus (BSMV)-mediated Bln1 or Bln2 overexpression increased susceptibility of barley to Bgh. Site-directed mutagenesis of Bln1 and Bln2, followed by Bimolecular fluorescence complementation (BiFC) assays signify that BLN1 and BLN2 interact with each other through an IQ binding domain, and can dimerize in planta. We then used BSMV-Induced Gene Silencing to knock down Bln1, followed by Barley1 GeneChip transcriptome analysis, to identify additional genes influenced by Bln1. Analysis of differential expression revealed a gene set enriched for those encoding proteins annotated to nuclear import and the secretory pathway, particularly Importin alpha 1-b, Nuclear transport factor 2, and Sec61 gamma subunit. Further functional analysis of these affected genes showed that when silenced, they too reduced barley susceptibility to Bgh. Taken together, we postulate that Bln1 is co-opted by Bgh to facilitate transport of effectors, influencing the establishment of Bgh compatibility on its barley host.