Xue-Cheng Zhang

Genome sequence of the palaeopolyploid soybean

Soybean (Glycine max) is one of the most important crop plants for seed protein and oil content, and for its capacity to fix atmospheric nitrogen through symbioses with soil-borne microorganisms. We sequenced the 1.1-gigabase genome by a whole-genome shotgun approach and integrated it with physical and high-density genetic maps to create a chromosome-scale draft sequence assembly. We predict 46,430 protein-coding genes, 70% more than Arabidopsis and similar to the poplar genome which, like soybean, is an ancient polyploid (palaeopolyploid). About 78% of the predicted genes occur in chromosome ends, which comprise less than one-half of the genome but account for nearly all of the genetic recombination. Genome duplications occurred at approximately 59 and 13 million years ago, resulting in a highly duplicated genome with nearly 75% of the genes present in multiple copies. The two duplication events were followed by gene diversification and loss, and numerous chromosome rearrangements. An accurate soybean genome sequence will facilitate the identification of the genetic basis of many soybean traits, and accelerate the creation of improved soybean varieties.

Molecular Evolution of Lysin Motif-Type Receptor-Like Kinases in Plants

The lysin motif (LysM) domain is an ancient and ubiquitous protein module that binds peptidoglycan and structurally related molecules. A genomic survey in a large number of species spanning all kingdoms reveals that the combination of LysM and receptor kinase domains is present exclusively in plants. However, the particular biological functions and molecular evolution of this gene family remain largely unknown. We show that LysM domains in plant LysM proteins are highly diversified and that a minimum of six distinct types of LysM motifs exist in plant LysM kinase proteins and five additional types of LysM motifs exist in nonkinase plant LysM proteins. Further, motif similarities suggest that plant LysM motifs are ancient. Although phylogenetic signals are not sufficient to resolve the earliest relationships, plant LysM motifs may have arisen through common ancestry with LysM motifs in other kingdoms. Within plants, the gene family has evolved through local and segmental duplications. The family has undergone further duplication and diversification in legumes, where some LysM kinase genes function as receptors for bacterial nodulation factor. Two pairs of homeologous regions were identified in soybean (Glycine max) based on microsynteny and fluorescence in situ hybridization. Expression data show that most plant LysM kinase genes are expressed predominantly in the root and that orthologous LysM kinase genes share similar tissue expression patterns. We also examined synteny around plant LysM kinase genes to help reconstruct scenarios for the evolution of this important gene family.

RPS4 -Mediated Disease Resistance Requires the Combined Presence of RPS4 Transcripts with Full-Length and Truncated Open Reading Frames

Arabidopsis RPS4 belongs to the Toll/interleukin-1 receptor (TIR)–nucleotide binding site (NBS)–Leu-rich repeat (LRR) class of disease resistance (R) genes. Like other family members in different plant species, RPS4 produces alternative transcripts with truncated open reading frames. The dominant alternative RPS4 transcripts are generated by retention of intron 3 or introns 2 and 3, which contain in-frame stop codons and lie downstream of the NBS-encoding exon. We analyzed the biological significance of these alternative transcripts in disease resistance by removing introns 2 and 3, either individually or in combination, from a functional RPS4-Ler (Landsberg erecta) transgene. Removal of one or both introns abolished the function of the RPS4 transgene, whereas expression was not affected. In addition, a truncated RPS4-Ler transgene encoding the putative TIR and NBS domains was not sufficient to confer resistance, suggesting that the combined presence of regular and alternative RPS4 transcripts is necessary for function. Interestingly, we observed partial resistance in transgenic lines expressing both intron-deficient and truncated transgenes. This finding confirms the requirement for regular and alternative RPS4 transcripts and indicates that alternative transcripts function at the protein level rather than as regulatory RNAs. Together with published results on the tobacco N gene, our data suggest that the generation of alternative TIR-NBS-LRR R gene transcripts is of general biological significance across plant species.

Alternative Splicing and mRNA Levels of the Disease Resistance Gene RPS4 Are Induced during Defense Responses

The Arabidopsis (Arabidopsis thaliana) disease resistance protein RESISTANCE TO PSEUDOMONAS SYRINGAE4 (RPS4) activates defenses in response to bacterial pathogens expressing avrRps4 in a gene-for-gene specific manner. The RPS4 gene produces multiple transcripts via alternative splicing of two regular introns flanking exon 3 and a cryptic intron within exon 3. We showed previously that RPS4-mediated resistance requires the combined presence of transcripts encoding both full-length and truncated open reading frames. Here, we demonstrate that alternative splicing of RPS4 undergoes dynamic changes specifically during the resistance response. Furthermore, RPS4 expression was induced by the presence of AvrRps4 in an EDS1-dependent manner. Interestingly, inducible alternative splicing was not limited to the avrRps4-RPS4 interaction, indicating that regulation of alternative splicing may be a general response to prime the plant stress response system. Intron-deficient transgenes lacking only one intron were previously shown to be nonfunctional. Here, we establish quantitatively that the absence of one intron had no effect on the splicing frequency of remaining introns. Given the lack of functionality of single intron-deficient transgenes, this suggests that the products of individual transcripts have distinct functions during RPS4-triggered resistance. Transient expression of truncated RPS4 proteins in Nicotiana benthamiana induced hypersensitive response-like cell death in the absence of AvrRps4. Interestingly, different truncated proteins had markedly differing stability. In summary, RPS4 function is regulated at multiple levels, including gene expression, alternative splicing, and protein stability, presumably to fine-tune activity and limit damage inflicted by activated RPS4 protein.

LYK4, a Lysin Motif Receptor-Like Kinase, Is Important for Chitin Signaling and Plant Innate Immunity in Arabidopsis

Chitin is commonly found in fungal cell walls and is one of the well-studied microbe/pathogen-associated molecular patterns. Previous studies showed that lysin motif (LysM)-containing proteins are essential for plant recognition of chitin, leading to the activation of plant innate immunity. In Arabidopsis (Arabidopsis thaliana), the LYK1/CERK1 (for LysM-containing receptor-like kinase1/chitin elicitor receptor kinase1) was shown to be essential for chitin recognition, whereas in rice (Oryza sativa), the LysM-containing protein, CEBiP (for chitin elicitor-binding protein), was shown to be involved in chitin recognition. Unlike LYK1/CERK1, CEBiP lacks an intracellular kinase domain. Arabidopsis possesses three CEBiP-like genes. Our data show that mutations in these genes, either singly or in combination, did not compromise the response to chitin treatment. Arabidopsis also contains five LYK genes. Analysis of mutations in LYK2, -3, -4, or -5 showed that LYK4 is also involved in chitin signaling. The lyk4 mutants showed reduced induction of chitin-responsive genes and diminished chitin-induced cytosolic calcium elevation as well as enhanced susceptibility to both the bacterial pathogen Pseudomonas syringae pv tomato DC3000 and the fungal pathogen Alternaria brassicicola, although these phenotypes were not as dramatic as that seen in the lyk1/cerk1 mutants. Similar to LYK1/CERK1, the LYK4 protein was also localized to the plasma membrane. Therefore, LYK4 may play a role in the chitin recognition receptor complex to assist chitin signal transduction and plant innate immunity.

Evolutionary genomics of LysM genes in land plants.

BackgroundThe ubiquitous LysM motif recognizes peptidoglycan, chitooligosaccharides (chitin) and, presumably, other structurally-related oligosaccharides. LysM-containing proteins were first shown to be involved in bacterial cell wall degradation and, more recently, were implicated in perceiving chitin (one of the established pathogen-associated molecular patterns) and lipo-chitin (nodulation factors) in flowering plants. However, the majority of LysM genes in plants remain functionally uncharacterized and the evolutionary history of complex LysM genes remains elusive.ResultsWe show that LysM-containing proteins display a wide range of complex domain architectures. However, only a simple core architecture is conserved across kingdoms. Each individual kingdom appears to have evolved a distinct array of domain architectures. We show that early plant lineages acquired four characteristic architectures and progressively lost several primitive architectures. We report plant LysM phylogenies and associated gene, protein and genomic features, and infer the relative timing of duplications of LYK genes.ConclusionWe report a domain architecture catalogue of LysM proteins across all kingdoms. The unique pattern of LysM protein domain architectures indicates the presence of distinctive evolutionary paths in individual kingdoms. We describe a comparative and evolutionary genomics study of LysM genes in plant kingdom. One of the two groups of tandemly arrayed plant LYK genes likely resulted from an ancient genome duplication followed by local genomic rearrangement, while the origin of the other groups of tandemly arrayed LYK genes remains obscure. Given the fact that no animal LysM motif-containing genes have been functionally characterized, this study provides clues to functional characterization of plant LysM genes and is also informative with regard to evolutionary and functional studies of animal LysM genes.

Chitin signaling and plant disease resistance

Chitin, a polymer of N-acetyl-D-glucosamine, is a component of the fungal cell wall and is not found in plants. Plant cells are equipped with chitin degrading enzymes to digest fungal cell walls and are capable of perceiving chitin fragments (chitooligosaccharides) released from fungal cell walls during fungal infection. Chitin recognition results in the activation of defense signaling pathways. Although chitin is a well recognized pathogen-associated molecular pattern (PAMPs), little is known about the molecular mechanism of chitin signaling. Recent studies identified a number of critical components in the chitin-elicited signaling pathway including a potential receptor, MAPK cascade and transcription factor network. Interestingly, the chitin signaling pathway overlaps with the phytobacterial flagellin- and EF-Tu-elicited signaling pathways, suggesting that plant cells may perceive different PAMPs from various pathogens via specialized receptors and then utilize a conserved, common downstream pathway to mediate disease resistance. Given the fact that fungal pathogens are major problems in many agricultural systems, research on chitin signaling could have significance to sustainable agriculture and biofuel and biomaterial production. Addendum to: Wan J, Zhang XC, Neece D, Ramonell KM, Clough S, Kim SY, Stacey MG, Stacey G.A LysM receptor-like kinase plays a critical role in Chitin signaling and fungal resistance in Arabidopsis. Plant Cell 2008; In press.

A member of the highly conserved FWL (tomato FW2.2-like) gene family is essential for soybean nodule organogenesis

A soybean homolog of the tomato FW2.2 gene, here named GmFWL1 (Glycine max FW2.2-like 1), was found to respond strongly to inoculation with the nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum. In tomato, the FW2.2 gene is hypothesized to control 30% of the variance in fruit weight by negatively regulating cell division. In the present study, the induction of GmFWL1 expression in root hair cells and nodules in response to B. japonicum inoculation was documented using quantitative RT-PCR and transcriptional fusions to both beta-glucuronidase (GUS) and green fluorescent protein (GFP). RNAi-mediated silencing of GmFWL1 expression resulted in a significant reduction in nodule number, with a concomitant reduction in nuclear size and changes in chromatin structure. The reduction in nuclear size is probably due to a change in DNA heterochromatinization, as the ploidy level of wild-type and RNAi-silenced nodule cells was similar. GmFWL1 was localized to the plasma membrane. The data suggest that GmFWL1 probably acts indirectly, perhaps through a cellular cascade, to affect chromatin structure/nuclei architecture. As previously proposed in tomato, this function may be a result of effects on plant cell division.

Activity-Based Metagenomic Screening and Biochemical Characterization of Bovine Ruminal Protozoan Glycoside Hydrolases

ABSTRACT The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.

Electrophysiological characterization of the arabidopsis avrRpt2-specific hypersensitive response in the absence of other bacterial signals

The hypersensitive response (HR) is defined as rapid cell collapse at the infection site and often accompanies plant resistance. The physiological processes leading to HR are not well understood. Here, we report an electrophysiological characterization of bacterial HR caused by a single avirulence gene in the absence of other bacterial signals. We used dexamethasone (dex)-inducible transgenic Arabidopsis (Arabidopsis thaliana) plants containing the avrRpt2 gene from Pseudomonas syringae pv tomato. Membrane depolarization in these plants began 1 to 1.5 h after dex application, hours before electrolyte leakage. Progressive depolarization was a sensitive early indicator of HR that occurred only in Arabidopsis leaf cells expressing both avrRpt2 and a functional RPS2 gene. Hyperpolarization of fully depolarized membranes by fusicoccin, a fungal toxin that activates the H+-ATPase, indicates that depolarization did not result from a nonfunctional pump or leaky membranes. Depolarization and electrolyte leakage were inhibited in RPS2 plants by the calcium channel blocker LaCl3, highly correlating these events and suggesting that Ca2+ entry into cells is required for both. Also correlated were inhibition of depolarization, electrolyte leakage, and HR following salicylic acid pretreatment. In salicylic acid-pretreated RPS2 seedlings, avrRpt2 transcript was produced after dex treatment. However, AvrRpt2 protein accumulation was greatly reduced, suggesting a possible mechanism for inhibition of HR in plants with induced resistance. This experimental system is a very sensitive assay that lends itself to the dissection of physiological processes leading to HR in plants, and provides a baseline for future research within a genetic framework.