Bojun Ma

Proteomic analysis of a disease-resistance-enhanced lesion mimic mutant spotted leaf 5 in rice.

BackgroundA lesion-mimic mutant in rice (Oryza sativa L.), spotted leaf 5 (spl5), displays a disease-resistance-enhanced phenotype, indicating that SPL5 negatively regulates cell death and resistance responses. To understand the molecular mechanisms of SPL5 mutation-induced cell death and resistance responses, a proteomics-based approach was used to identify differentially accumulated proteins between the spl5 mutant and wild type (WT).ResultsProteomic data from two-dimensional gel electrophoresis showed that 14 candidate proteins were significantly up- or down-regulated in the spl5 mutant compared with WT. These proteins are involved in diverse biological processes including pre-mRNA splicing, amino acid metabolism, photosynthesis, glycolysis, reactive oxygen species (ROS) metabolism, and defense responses. Two candidate proteins with a significant up-regulation in spl5 – APX7, a key ROS metabolism enzyme and Chia2a, a pathogenesis-related protein – were further analyzed by qPCR and enzyme activity assays. Consistent with the proteomic results, both transcript levels and enzyme activities of APX7 and Chia2a were significantly induced during the course of lesion formation in spl5 leaves.ConclusionsMany functional proteins involving various metabolisms were likely to be responsible for the lesion formation of spl5 mutant. Generally, in spl5, the up-regulated proteins involve in defense response or PCD, and the down-regulated ones involve in amino acid metabolism and photosynthesis. These results may help to gain new insight into the molecular mechanism underlying spl5-induced cell death and disease resistance in plants.

Clathrin Light Chains Regulate Clathrin-Mediated Trafficking, Auxin Signaling, and Development in Arabidopsis

Clathrin-mediated endocytosis of the plasma membrane proteins is regulated by auxin and the extracellular auxin receptor ABP1. This work demonstrates that clathrin light chains are key regulators of clathrin heavy chain membrane localization and auxin-dependent clathrin-mediated trafficking from the plasma membrane and trans-Golgi network/early endosome downstream of ABP1-mediated signaling. Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development.

Identification and characterization of putative CIPK genes in maize.

Calcium (Ca) plays a crucial role as a second messenger in intracellular signaling elicited by developmental and environmental cues. Calcineurin B-like proteins (CBLs) and their target proteins, CBL-interacting protein kinases (CIPKs) have emerged as a key Ca(2+)-mediated signaling network in response to stresses in plants. Bioinformatic analysis was used to identify 43 putative ZmCIPK (Zea mays CIPK) genes in the genome of maize inbred line B73. Based on gene structures, these ZmCIPKs were divided into intron-rich and intron-poor groups. Phylogenetic analysis indicated that the ZmCIPK family had a high evolutionary relationship with the rice CIPK family of 30 members. Microarray data and RT-PCR assay showed that ZmCIPK genes transcriptionally responded to abiotic stresses, and that 24, 31, 20 and 19 ZmCIPK genes were up-regulated by salt, drought, heat and cold stresses, respectively. There were different expression patterns of ZmCIPKs between cold-tolerant inbred line B73 and cold-sensitive inbred line Mo17 under cold stress. Our findings will aid further molecular dissection of biological functions of the CIPKs in maize, and provide new insight into the CBL-CIPK signaling network in plants.

SPL5, a cell death and defense-related gene, encodes a putative splicing factor 3b subunit 3 (SF3b3) in rice

A lesion-mimic phenotype in rice (Oryza sativa L.) spotted leaf 5 (spl5) indicates that wild-type SPL5 negatively regulates cell death and resistance responses. Previously, the spl5 gene was already mapped to the 80-kb region between two markers SSR7 and RM7121 through a map-based cloning approach. Here, we further showed that the spl5 gene was delimitated into a 15.1-kb genomic region by the high-resolution sequence target site (STS) markers. Subsequent sequencing in this region of spl5 mutant revealed that one candidate gene harbored a single-base deletion, resulting in a frame-shift mutation and a premature stop codon. Bioinformatic analysis showed that SPL5 gene encodes a putative splicing factor 3b subunit 3 (SF3b3) and might be involved in splicing reactions of pre-mature RNAs participating in the regulation of cell death and resistance responses. Further analysis showed that wild-type SPL5 did functionally complement the spl5 phenotype. The data presented here clearly indicate that the SPL5 negatively regulates cell death and resistance responses via modulating RNA splicing in plants.

Transcriptome profiling of the spl5 mutant reveals that SPL5 has a negative role in the biosynthesis of serotonin for rice disease resistance

BackgroundRice mutant, spl5 (spotted leaf 5), has spontaneous hypersensitive-like lesions on its leaves and shows enhanced resistance to pathogens, indicating that SPL5 plays a role in programmed cell death (PCD) and disease resistance. To understand the molecular mechanism of SPL5 gene, we investigated the transcriptome profiles of the spl5 mutant leaves with few lesions (FL) and leaves with many lesions (ML) compared to the wild-type (WT) leaves respectively by microarray.ResultsThe data from microarray revealed that 243 and 896 candidate genes (Fold change ≥ 3.0) were up- or down-regulated in the spl5-FL and spl5-ML, respectively, and a large number of these genes involved in biotic defense responses or reactive oxygen species (ROS) metabolism. Interestingly, according to our microarray and real-time PCR assays, the expressions of a transcription factor OsWRKY14 and genes responsible for the biosynthesis of serotonin, anthranilate synthase (AS), indole-3-glycerolphosphate synthase (IGPS), tryptophan synthase (TS) and tryptophan decarboxylase (TDC) were significantly up-regulated in the spl5 mutant. It has been reported previously that TS and TDC expressions are regulated by OsWRKY14 in rice, which raises the possibility that OsWRKY14 regulates serotonin production through the up-regulation of TS and TDC. Our HPLC analysis further confirmed that serotonin levels were higher in the leaves of spl5 mutant than that in WT.ConclusionsSince the serotonin plays a critical role in inducing disease-resistance, the increased serotonin level may contribute, at least partly, to the disease resistance in spl5. The SPL5 gene may act as a negative regulatory factor activating the serotonin metabolic pathway, and these results might provide a new insight into the spl5-induced defense response mechanisms in plants.

ChIP-Seq: A Powerful Tool for Studying Protein-DNA Interactions in Plants

DNA-binding proteins, including transcription factors, epigenetic and chromatin modifiers, control gene expressions in plants. To pinpoint the binding sits of DNA-binding proteins in genome is crucial for decoding gene regulatory networks. Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-Seq) is a widely used approach to identify the DNA regions bound by a specific protein in vivo. The information generated from ChIP-Seq has tremendously advanced our understanding on the mechanism of transcription factors, cofactors and histone modifications in regulating gene expression. In this review, we reviewed the recent research advance of ChIP-Seq in plants, including description of the ChIP-Seq workflow and its various applications in plants, and in addition, provided perspective of the potential advances of ChIP-Seq.

An efficient CAPS marker for bacterial-blight resistance gene xa25 in rice

Abstract The recessive Xanthomonas oryzae pv. oryzae (Xoo) resistance gene, xa25, which mediates race-specific resistance to the Xoo isolate PXO339, has previously been characterized in rice (Oryza sativa L.). Its dominant allele, Xa25, is transcriptionally induced by Xoo infection, which in turn results in susceptibility to PXO339. Utilizing a base substitution (−40, G to T) in the xa25/Xa25 promoters, a cleaved amplified polymorphic sequence (CAPS) marker was designed for effective identification of the xa25 gene. This marker co-segregated with the xa25 gene and explicitly distinguished the genotypes of xa25/Xa25 gene in rice. The marker was further tested on nine Chinese rice landraces, revealing the association of xa25/Xa25 genotypes with resistance/susceptibility to PXO339, respectively. Therefore, this marker is reliable and cost-effective for marker-assisted selection of the xa25 gene in rice.