Gautam Shirsekar

The Magnaporthe oryzae Effector AvrPiz-t Targets the RING E3 Ubiquitin Ligase APIP6 to Suppress Pathogen-Associated Molecular Pattern-Triggered Immunity in Rice

This work shows that the Magnaporthe oryzae effector AvrPiz-t enters into rice cells to target the RING E3 ubiquitin ligase APIP6 for suppression of PAMP-triggered immunity in rice. It also describes that APIP6 degrades AvrPiz-t in planta and positively regulates basal defense to M. oryzae. Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants.

The U-Box E3 Ligase SPL11/PUB13 Is a Convergence Point of Defense and Flowering Signaling in Plants

Plants use the ubiquitin-proteasome system (UPS) to regulate nearly every aspect of growth and development and to respond to abiotic and biotic stresses. Among the three major enzymes involved in the UPS, E3 ligases determine substrate specificity and actively participate in many biological processes in plants. Emerging evidence shows that some E3 ligases have multiple functions and serve as a connection node in plant signaling. Here, we review the dual functions of the U-box and armadillo (ARM) repeat domain E3 ligase SPL11 in rice and of its ortholog PUB13 in Arabidopsis in modulating innate immunity and flowering. Both SPL11 and PUB13 negatively regulate programmed cell death (PCD) and defense. Intriguingly, SPL11 promotes flowering under long-day (LD) conditions in rice while PUB13 suppresses flowering under LD conditions in Arabidopsis. SPL11 regulates defense through a putative GAP protein and regulates flowering through an RNA-binding protein. PUB13 modulates defense through FLS2 and may control flowering through HFR1. Moreover, PUB13-mediated defense and flowering depend on the plant hormone salicylic acid (SA). The similar functions of SPL11 and PUB13, and the complementation of the pub13 cell death and flowering phenotypes by Spl11 indicate that Spl11/PUB13 is an ancient, functionally conserved locus in monocot and dicot plants. In the process of speciation, the downstream signaling components have, however, diversified in these two species. We conclude by proposing working models of how SPL11 and PUB13 and their associated proteins modulate both defense and flowering in monocot and dicot plants.

The E3 Ligase APIP10 Connects the Effector AvrPiz-t to the NLR Receptor Piz-t in Rice

Although nucleotide-binding domain, leucine-rich repeat (NLR) proteins are the major immune receptors in plants, the mechanism that controls their activation and immune signaling remains elusive. Here, we report that the avirulence effector AvrPiz-t from Magnaporthe oryzae targets the rice E3 ligase APIP10 for degradation, but that APIP10, in return, ubiquitinates AvrPiz-t and thereby causes its degradation. Silencing of APIP10 in the non-Piz-t background compromises the basal defense against M. oryzae. Conversely, silencing of APIP10 in the Piz-t background causes cell death, significant accumulation of Piz-t, and enhanced resistance to M. oryzae, suggesting that APIP10 is a negative regulator of Piz-t. We show that APIP10 promotes degradation of Piz-t via the 26S proteasome system. Furthermore, we demonstrate that AvrPiz-t stabilizes Piz-t during M. oryzae infection. Together, our results show that APIP10 is a novel E3 ligase that functionally connects the fungal effector AvrPiz-t to its NLR receptor Piz-t in rice.

Role of Ubiquitination in Plant Innate Immunity and Pathogen Virulence

Plant diseases are a major constraint for stable crop production in the world. Plants are constantly threatened by different pathogens and have developed an array of mechanisms to defend themselves. A growing body of evidence indicates that ubiquitination, which is one of the most important cellular processes for protein modification in eukaryotic organisms, is involved in the regulation of host defense signaling. Pathogens also exploit ubiquitination to block or interfere with plant defenses. Recent studies in a few model plants have demonstrated that ubiquitination plays a critical role in plant–pathogen interactions that lead either to plant resistance or to successful pathogen invasion of the plant host. This review discusses recent findings about the functions of ubiquitination in host defense and pathogen invasion.

Quantification of hydrogen peroxide in plant tissues using Amplex Red.

Reactive oxygen species (ROS) are by-products of photosynthesis and respiration in plant tissues. Abiotic and biotic stressors also induce the production and temporary accumulation of ROS in plants, including hydrogen peroxide (H2O2), whereby they can act as secondary messengers/chemical mediators in plant defense signaling and lead to programmed cell death. H2O2 acts as a hub for critical information flow in plants. Despite such key roles in fundamental cellular processes, reliable determination of H2O2 levels in plant tissues is hard to achieve. We optimized an Amplex Red-based quantitation method for H2O2 estimation from plant tissue lysate. The standard limit of detection and quantitation was determined as 6 and 18picomol respectively. In this study we also quantified constitutive and/or induced levels of H2O2 in three model plants, Pinus nigra (Austrian pine), Oryza sativa (rice), and Arabidopsis thaliana. Overall, assay sensitivity was in the nmolg-1 FW range. Commonly used additives for H2O2 extraction such as activated charcoal, ammonium sulfate, perchloric acid, polyvinylpolypyrrolidone, and trichloroacetic acid either degraded H2O2 directly or interfered with the Amplex Red assay. Finally, We measured stability of Amplex Red working solution over one month of storage at -80°C and found it to be significantly stable over time. With appropriate modifications, this optimized method should be applicable to any plant tissue.

Identification and Characterization of Suppressor Mutants of spl11-Mediated Cell Death in Rice

Lesion mimic mutants have been used to dissect programmed cell death (PCD) and defense-related pathways in plants. The rice lesion-mimic mutant spl11 exhibits race nonspecific resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae and the fungal pathogen Magnaporthe oryzae. Spl11 encodes an E3 ubiquitin ligase and is a negative regulator of PCD in rice. To study the regulation of Spl11-mediated PCD, we performed a genetic screen and identified three spl11 cell-death suppressor (sds) mutants. These suppressors were characterized for their resistance to X. oryzae pv. oryzae and M. oryzae and for their expression of defense-related genes. The suppression of the cell-death phenotypes was generally correlated with reduced expression of defense-related genes. When rice was challenged with avirulent isolates of M. oryzae, the disease phenotype was unaffected in the sds mutants, indicating that the suppression might be Spl11-mediated pathway specific and may only be involved in basal defense. In addition, we mapped one of the suppressor mutations to a 140-kb interval on the long arm of rice chromosome 1. Identification and characterization of these sds mutants should facilitate our efforts to elucidate the Spl11-mediated PCD pathway.

OsWRKY67 Plays a Positive Role in Basal and XA21-Mediated Resistance in Rice

WRKY proteins play important roles in transcriptional reprogramming in plants in response to various stresses including pathogen attack. In this study, we functionally characterized a rice WRKY gene, OsWRKY67, whose expression is upregulated against pathogen challenges. Activation of OsWRKY67 by T-DNA tagging significantly improved the resistance against two rice pathogens, Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo). Reactive oxygen species (ROS) rapidly accumulated in OsWRKY67 activation mutant lines in response to elicitor treatment, compared with the controls. Overexpression of OsWRKY67 in rice confirmed enhanced disease resistance, but led to a restriction of plant growth in transgenic lines with high levels of OsWRKY67 protein. OsWRKY67 RNAi lines significantly reduced resistance to M. oryzae and Xoo isolates tested, and abolished XA21-mediated resistance, implying the possibility of broad-spectrum resistance from OsWRKY67. Transcriptional activity and subcellular localization assays indicated that OsWRKY67 is present in the nucleus where it functions as a transcriptional activator. Quantitative PCR revealed that the pathogenesis-related genes, PR1a, PR1b, PR4, PR10a, and PR10b, are upregulated in OsWRKY67 overexpression lines. Therefore, these results suggest that OsWRKY67 positively regulates basal and XA21-mediated resistance, and is a promising candidate for genetic improvement of disease resistance in rice.

Validation of race non-specific, adult plant leaf rust resistance genes Lr34 and Lr67 in some common wheat (Triticum aestivum L.) cultivars

Twelve common wheat cultivars with Lr34 postulated based on leaf tip necrosis and other observations were studied for adult plant resistance to leaf rust (Puccinia triticina) pathotype 77-5 (121R63-1), and validation of Lr34 and Lr67 through allelic tests. Line 897 and Frontana, known Lr34 carriers, served as checks. Resistance was governed by one dominant gene each in C 306, Kalyansona, Line 897, NI 5439 and WH 147; and by two dominant genes each in Frontana, GW 173, HD 2189, HI 1077, HP 1744, K 9107, PBW 175, PBW 373, and UP 2338. Allelic tests showed presence of Lr34 in Frontana, GW 173, HD 2189, HP 1744 and Line 897; and of Lr67 in C 306, NI 5439 and K 9107. Comparison is drawn with reports based on molecular markers. Our findings should be useful in multiple disease resistance breeding as Lr34 and Lr67 are associated with resistance to multiple wheat pathogens.