Liangying Dai

The Broad-Spectrum Blast Resistance Gene Pi9 Encodes a Nucleotide-Binding Site–Leucine-Rich Repeat Protein and Is a Member of a Multigene Family in Rice

The broad-spectrum rice blast resistance gene Pi9 was cloned using a map-based cloning strategy. Sequencing of a 76-kb bacterial artificial chromosome (BAC) contig spanning the Pi9 locus led to identification of six tandemly arranged resistance-like genes with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) (Nbs1-Pi9–Nbs6-Pi9). Analysis of selected Pi9 deletion mutants and transformation of a 45-kb fragment from the BAC contig into the susceptible rice cultivar TP309 narrowed down Pi9 to the candidate genes Nbs2-Pi9 and Nbs3-Pi9. Disease evaluation of the transgenic lines carrying the individual candidate genes confirmed that Nbs2-Pi9 is the Pi9 gene. Sequence comparison analysis revealed that the six paralogs at the Pi9 locus belong to four classes and gene duplication might be one of the major evolutionary forces contributing to the formation of the NBS–LRR gene cluster. Semiquantitative reverse transcriptase (RT)–PCR analysis showed that Pi9 was constitutively expressed in the Pi9-resistant plants and was not induced by blast infection. The cloned Pi9 gene provides a starting point to elucidate the molecular basis of the broad-spectrum disease resistance and the evolutionary mechanisms of blast resistance gene clusters in rice.

Recent progress and understanding of the molecular mechanisms of the rice–Magnaporthe oryzae interaction

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is the most devastating disease of rice and severely affects crop stability and sustainability worldwide. This disease has advanced to become one of the premier model fungal pathosystems for host-pathogen interactions because of the depth of comprehensive studies in both species using modern genetic, genomic, proteomic and bioinformatic approaches. Many fungal genes involved in pathogenicity and rice genes involved in effector recognition and defence responses have been identified over the past decade. Specifically, the cloning of a total of nine avirulence (Avr) genes in M. oryzae, 13 rice resistance (R) genes and two rice blast quantitative trait loci (QTLs) has provided new insights into the molecular basis of fungal and plant interactions. In this article, we consider the new findings on the structure and function of the recently cloned R and Avr genes, and provide perspectives for future research directions towards a better understanding of the molecular underpinnings of the rice-M. oryzae interaction.

Recent Progress in Elucidating the Structure, Function and Evolution of Disease Resistance Genes in Plants

Plants employ multifaceted mechanisms to fight with numerous pathogens in nature. Resistance (R) genes are the most effective weapons against pathogen invasion since they can specifically recognize the corresponding pathogen effectors or associated protein(s) to activate plant immune responses at the site of infection. Up to date, over 70 R genes have been isolated from various plant species. Most R proteins contain conserved motifs such as nucleotide-binding site (NBS), leucine-rich repeat (LRR), Toll-interleukin-1 receptor domain (TIR, homologous to cytoplasmic domains of the Drosophila Toll protein and the mammalian interleukin-1 receptor), coiled-coil (CC) or leucine zipper (LZ) structure and protein kinase domain (PK). Recent results indicate that these domains play significant roles in R protein interactions with effector proteins from pathogens and in activating signal transduction pathways involved in innate immunity. This review highlights an overview of the recent progress in elucidating the structure, function and evolution of the isolated R genes in different plant-pathogen interaction systems.

The U-Box/ARM E3 Ligase PUB13 Regulates Cell Death, Defense, and Flowering Time in Arabidopsis

The components in plant signal transduction pathways are intertwined and affect each other to coordinate plant growth, development, and defenses to stresses. The role of ubiquitination in connecting these pathways, particularly plant innate immunity and flowering, is largely unknown. Here, we report the dual roles for the Arabidopsis (Arabidopsis thaliana) Plant U-box protein13 (PUB13) in defense and flowering time control. In vitro ubiquitination assays indicated that PUB13 is an active E3 ubiquitin ligase and that the intact U-box domain is required for the E3 ligase activity. Disruption of the PUB13 gene by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. The cell death, hydrogen peroxide accumulation, and resistance to necrotrophic pathogens in pub13 are enhanced when plants are pretreated with high humidity. Importantly, pub13 also shows early flowering under middle- and long-day conditions, in which the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and FLOWERING LOCUS T is induced while FLOWERING LOCUS C expression is suppressed. Finally, we found that two components involved in the SA-mediated signaling pathway, SID2 and PAD4, are required for the defense and flowering-time phenotypes caused by the loss of function of PUB13. Taken together, our data demonstrate that PUB13 acts as an important node connecting SA-dependent defense signaling and flowering time regulation in Arabidopsis.

The SINA E3 Ligase OsDIS1 Negatively Regulates Drought Response in Rice

Ubiquitin-regulated protein degradation is a critical regulatory mechanism that controls a wide range of biological processes in plants. Here, we report that OsDIS1 (for Oryza sativa drought-induced SINA protein 1), a C3HC4 RING finger E3 ligase, is involved in drought-stress signal transduction in rice (O. sativa). The expression of OsDIS1 was up-regulated by drought treatment. In vitro ubiquitination assays showed that OsDIS1 possessed E3 ubiquitin ligase activity and that the conserved region of the RING finger was required for the activity. Transient expression assays in Nicotiana benthamiana leaves and rice protoplasts indicated that OsDIS1 was localized predominantly in the nucleus. Overexpression of OsDIS1 reduced drought tolerance in transgenic rice plants, while RNA interference silencing of OsDIS1 enhanced drought tolerance. Microarray analysis revealed that a large number of drought-responsive genes were induced or suppressed in the OsDIS1 overexpression plants under normal and drought conditions. Yeast two-hybrid screening showed that OsDIS1 interacted with OsNek6 (for O. sativa NIMA-related kinase 6), a tubulin complex-related serine/threonine protein kinase. Coexpression assays in N. benthamiana leaves indicated that OsNek6 was degraded by OsDIS1 via the 26S proteasome-dependent pathway and that this degradation was abolished by the OsDIS1(H71Y) mutation, which is essential for its E3 ligase activity. Together, these results demonstrate that OsDIS1 plays a negative role in drought stress tolerance through transcriptional regulation of diverse stress-related genes and possibly through posttranslational regulation of OsNek6 in rice.

GmCOI1, a Soybean F-Box Protein Gene, Shows Ability to Mediate Jasmonate-Regulated Plant Defense and Fertility in Arabidopsis

The F-box protein gene COI1 from Arabidopsis plays a fundamental role in response to jasmonates, which regulate plant root growth, pollen fertility, wounding and healing, and defense against pathogens and insects. Null mutations in COI1 were previously found to abolish all the jasmonate responses, and the Arabidopsis coil-1 mutant is male sterile and susceptible to pathogen infection. In this study, we isolated an F-box protein gene from soybean, which shares significant homology with the Arabidopsis COI1 and similarly contains an F-box motif and leucine rich repeats (LRR), here designated GmCOI1 (Glycine max L. (Merr.) COI1). To test whether the sequence homology and structural similarity are indicative of functional conservation, we expressed GmCOI1 in the Arabidopsis coil-1 mutant. The transgenic coil-1 plants with expression of the GmCOI1 gene were found to exhibit normal jasmonate responses, including jasmonate-regulated plant defense and fertility. In addition, the chimerical proteins with swapped domain of the F-box motif or LRR between GmCOI1 and COI1 were shown to functionally complement the coil-1 mutation. Furthermore, GmCOI1 was found to assemble into the Skpl-Cullin-F-box (SCF) complexes, similar to the formation of the Arabidopsis SCF(COO1). These data demonstrate the soybean F-box protein gene GmCOI1 is able to mediate jasmonate-regulated plant defense and fertility in Arabidopsis, which implies a generic jasmonate pathway with conserved signal components in different plant species.

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.

Molecular Mapping of the New Blast Resistance Genes Pi47 and Pi48 in the Durably Resistant Local Rice Cultivar Xiangzi 3150

The indica rice cultivar Xiangzi 3150 (XZ3150) confers a high level of resistance to 95% of the isolates of Magnaporthe oryzae (the agent of rice blast disease) collected in Hunan Province, China. To identify the resistance (R) gene(s) controlling the high level of resistance in this cultivar, we developed 286 F(9) recombinant inbred lines (RILs) from a cross between XZ3150 and the highly susceptible cultivar CO39. Inoculation of the RILs and an F(2) population from a cross between the two cultivars with the avirulent isolate 193-1-1 in the growth chamber indicated the presence of two dominant R genes in XZ3150. A linkage map with 134 polymorphic simple sequence repeat and single feature polymorphism markers was constructed with the genotype data of the 286 RILs. Composite interval mapping (CIM) using the results of 193-1-1 inoculation showed that two major R genes, designated Pi47 and Pi48, were located between RM206 and RM224 on chromosome 11, and between RM5364 and RM7102 on chromosome 12, respectively. Interestingly, the CIM analysis of the four resistant components of the RILs to the field blast population revealed that Pi47 and Pi48 were also the major genetic factors responsible for the field resistance in XZ3150. The DNA markers linked to the new R genes identified in this study should be useful for further fine mapping, gene cloning, and marker-aided breeding of blast-resistant rice cultivars.

Quantitative trait loci associated with seed set under high temperature stress at the flowering stage in rice (Oryza sativa L.)

High temperature stress (HTS), an increasingly important problem in rice production, significantly reduces rice yield by reducing seed set percentage (SSP). Breeding rice varieties with tolerance to HTS at the flowering stage is therefore essential for maintaining rice production as the climate continues to warm. In this study, two quantitative trait loci (QTL) underlying tolerance to HTS were identified using the recombinant inbred lines (RILs) derived from a cross between the HTS-tolerant rice cultivar 996 and the sensitive cultivar 4628. SSP was used as the heat-tolerance indicator for the lines, which were subjected to HTS at the flowering stage in both field and growth chamber experiments. Two major QTL that affected SSP in both conditions were detected in the interval between RM5687 and RM471 on chromosome 4, and between RM6132 and RM6100 on chromosome 10. The QTL located on chromosome 4 explained 21.3% in field and 25.8% in growth chamber of the total phenotypic variation in SSP, and increased the SSP of plants subjected to HTS by 9.1% in field and by 9.3% in growth chamber. The second QTL located on chromosome 10 explained 11.5% in field and 11.6% in growth chamber of the total phenotypic variation in SSP, and increased the SSP of plants subjected to HTS by 7.2% in field and 7.0% in growth chamber. The positive additive effects of the two QTL were derived from the 996 alleles. The two major QTL identified in this study could be useful for further fine mapping and cloning of these genes and for molecular marker-aided breeding of heat-tolerant rice cultivars.

Genomic structure and evolution of the Pi2/9 locus in wild rice species

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a devastating disease of rice worldwide. Among the 85 mapped resistance (R) genes against blast, 13 have been cloned and characterized. However, how these genes originated and how they evolved in the Oryza genus remains unclear. We previously cloned the rice blast R-genes Pi2, Pi9, and Piz-t, and analyzed their genomic structure and evolution in cultivated rice. In this study, we determined the genomic sequences of the Pi2/9 locus in four wild Oryza species representing three genomes (AA, BB and CC). The number of Pi2/9 family members in the four wild species ranges from two copies to 12 copies. Although these genes are conserved in structure and categorized into the same subfamily, sequence duplications and subsequent inversions or uneven crossing overs were observed, suggesting that the locus in different wild species has undergone dynamic changes. Positive selection was found in the leucine-rich repeat region of most members, especially in the largest clade where Pi9 is included. We also provide evidence that the Pi9 gene is more related to its homologues in the recurrent line and other rice cultivars than to those in its alleged donor species O. minuta, indicating a possible origin of the Pi9 gene from O. sativa. Comparative sequence analysis between the four wild Oryza species and the previously established reference sequences in cultivated rice species at the Pi2/9 locus has provided extensive and unique information on the genomic structure and evolution of a complex R-gene cluster in the Oryza genus.