Plant WRKY transcription factors act as either positive or negative regulators of plant basal disease resistance. To comprehensively characterise the complicated functional network, we isolated OsWRKY77 from rice seedlings treated with salicylic acid. OsWRKY77 is a typical WRKY transcription factor, based on in its protein structure analysis, nuclear localisation of the fused OsWRKY77-GFP protein and gel electrophoretic mobility shift assay binding, which demonstrated that OsWRKY77 was able to bind a W-box. Transgenic Arabidopsis lines overexpressing OsWRKY77 repressed growth of Pseudomonas syringae pv. tomato DC3000 (PstDC300), and the reduced susceptibility was associated with enhanced expression of defence-related PR1, PR2 and PR5 genes. These results show that OsWRKY77 is a positive regulator of PR gene expression and basal resistance to the bacterial pathogen PstDC3000.

The prevalence of de novo coding genes is controversial due to length and coding constraints. Noncoding genes, especially small ones, are freer to evolve de novo by comparison. The best examples are microRNAs (miRNAs), a large class of regulatory molecules ∼22 nt in length. Here, we study six de novo miRNAs in , which, like most new genes, are testis-specific. We ask how and why de novo genes die because gene death must be sufficiently frequent to balance the many new births. By knocking out each miRNA gene, we analyzed their contributions to the nine components of male fitness (sperm production, length, and competitiveness, among others). To our surprise, the knockout mutants often perform better than the wild type in some components, and slightly worse in others. When two of the younger miRNAs are assayed in long-term laboratory populations, their total fitness contributions are found to be essentially zero. These results collectively suggest that adaptive de novo genes die regularly, not due to the loss of functionality, but due to the canceling out of positive and negative fitness effects, which may be characterized as "quasi-neutrality." Since de novo genes often emerge adaptively and become lost later, they reveal ongoing period-specific adaptations, reminiscent of the "Red-Queen" metaphor for long-term evolution.

Molecular evolution is believed to proceed in small steps. The step size can be defined by a distance reflecting physico-chemical disparities between amino acid (AA) pairs that can be exchanged by single 1 bp mutations. We show that AA substitution rates are strongly and negatively correlated with this distance but only when positive selection is relatively weak. We use the McDonald and Kreitman (MK) test to separate the influences of positive and negative selection. While negative selection is indeed stronger on AA substitutions generating larger changes in chemical properties of amino acids, positive selection operates by different rules. For 65 of the 75 possible pairs, positive selection is comparable in strength regardless of AA distance. However, the 10 pairs under the strongest positive selection all exhibit large leaps in chemical properties. Five of the 10 pairs are shared between hominoids and Drosophila, thus hinting at a common but modest biochemical basis of adaptation across taxa. The hypothesis that adaptive changes often take large functional steps will need to be extensively tested. If validated, molecular models will need to better integrate positive and negative selection in the search for adaptive signal.An opposite argument for large-step evolution can be stated as follows: Natural selection, either positive or negative, can "discern" large-step changes better than small-step ones. For example, replacing isoleucine (Ile) with the chemically similar Valine (Val) would not alter the protein structure as much as its replacement by Arginine (Arg), which is very different chemically. In this view, an Ile à Arg replacement may be either much worse, or much better, than the Ile à Val replacement. Therefore, while negative selection would accept small-step changes (e.g., Ile à Val), positive selection might in fact favor large-step ones (e.g., Ile à Arg). The conventional wisdom is a postulate, not a fact.Clearly, the arguments must be resolved by empirical means. The companion study (Chen and Wu) has shown that AA substitutions start to deviate from the small-step rule when negative selection becomes weaker and/or positive selection becomes stronger. In the same vein, this study aims to separate the two effects on the rate of AA substitutions.

The widely accepted view that evolution proceeds in small steps is based on two premises: 1) negative selection acts strongly against large differences and 2) positive selection favors small-step changes. The two premises are not biologically connected and should be evaluated separately. We now extend a previous approach to studying codon evolution in the entire genome. Codon substitution rate is a function of the physicochemical distance between amino acids (AAs), equated with the step size of evolution. Between nine pairs of closely related species of plants, invertebrates, and vertebrates, the evolutionary rate is strongly and negatively correlated with a set of AA distances (ΔU, scaled to [0, 1]). ΔU, a composite measure of evolutionary rates across diverse taxa, is influenced by almost all of the 48 physicochemical properties used here. The new analyses reveal a crucial trend hidden from previous studies: ΔU is strongly correlated with the evolutionary rate (R2 > 0.8) only when the genes are predominantly under negative selection. Because most genes in most taxa are strongly constrained by negative selection, ΔU has indeed appeared to be a nearly universal measure of codon evolution. In conclusion, molecular evolution at the codon level generally takes small steps due to the prevailing negative selection. Whether positive selection may, or may not, follow the small-step rule is addressed in a companion study.