Characterization of the small RNA transcriptome in plant-microbe (Brassica/Erwinia) interactions by high-throughput sequencing.

Non-coding, small RNAs (sRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans; however, the role and mechanisms of these sRNA in plant immunity is largely unknown. To determine possible roles of sRNA in plant-pathogen interaction, we carried out a high-throughput sRNA sequencing of Brassica campestris using non-infected plants and plants infected with Erwinia carotovora. Consistent with our hypothesis that distinct classes of host sRNAs alerts their expression levels in response to infection, we found that: (1) host 28-nt sRNAs were strongly increased under pathogen infection; and (2) a group of host sRNAs homologous to the pathogen genome also accumulated at significantly higher level. Our data thus suggest several distinct classes of the host sRNAs may enhance their function by up-regulation of their expression/stability in response to bacterial pathogen challenges.

[Epigenetics of plant vernalization regulated by non-coding RNAs].

Many higher plants must experience a period of winter cold to accomplish the transition from vegetative to reproductive growth. This biological process is called vernalization. Some crops such as wheat (Triticum aestivum L.) and oilseed rape (Brassica napus L.) produce seeds as edible organs, and therefore special measures of rotation and cultivation are necessary for plants to go through an early vernalization for flower differentiation and development, whereas the other crops such as Chinese cabbage (B rapa ssp. pekinenesis) and cabbage (Brassica napus L.) produce leafy heads as edible organs, and additional practice should be taken to avoid vernalization for a prolonged and fully vegetative growth. Before vernalization, flowering is repressed by the action of a gene called Flowering Locus C (FLC). This paper reviewed the function of non-coding RNAs and some proteins including VRN1, VRN2, and VIN3 in epigenetic regulation of FLC during vernalization.

A novel method for constructing pathogen-regulated small RNA cDNA library.

Pathogen-responsive endogenous small non-coding RNAs regulate gene expression in relation to plant immune responses by serving as RNA silencing machinery. Decay caused by the bacterium, Erwinia carotovora subsp. carotovora (Ecc), often leads to soft rot disease in the plant Brassica campestris L. ssp. pekinensis (Bcp). To discover endogenous small RNA species in Bcp in response to Ecc infection, we developed a highly efficient approach for cloning pathogen-regulated small RNAs. A group of degenerate stem-loop reverse primers was designed to synthesize first single-stranded cDNA (sscDNA) and the sscDNA was then tailed with a poly(C) at its 3' end to create a forward priming site. A novel cDNA/RNA subtractive hybridization was performed to capture Ecc-regulated small RNAs and this subsequently allowed construction of small RNA cDNA libraries for sequencing.

A multidrug resistance transporter in Magnaporthe is required for host penetration and for survival during oxidative stress.

In prokaryotes and eukaryotes, multidrug resistance (MDR) transporters use energy-dependent efflux action to regulate the intracellular levels of antibiotic or xenobiotic compounds. Using mutational analysis of ABC3, we define an important role for such MDR-based efflux during the host penetration step of Magnaporthe grisea pathogenesis. Mutants lacking ABC3 were completely nonpathogenic but were surprisingly capable of penetrating thin cellophane membranes to some extent. The inability of abc3Delta to penetrate the host surface was most likely a consequence of excessive buildup of peroxide and accumulation of an inhibitory metabolite(s) within the mutant appressoria. Treatment with antioxidants partially suppressed the host penetration defects in the abc3Delta mutant. abc3Delta was highly sensitive to oxidative stress and was unable to survive the host environment and invasive growth conditions. ABC3 transcript levels were redox-regulated, and on host surfaces, the activation of ABC3 occurred during initial stages of blast disease establishment. An Abc3-green fluorescent protein fusion localized to the plasma membrane in early appressoria (and in penetration hyphae) but became predominantly vacuolar during appressorial maturity. We propose that ABC3 function helps Magnaporthe to cope with cytotoxicity and oxidative stress within the appressoria during early stages of infection-related morphogenesis and likely imparts defense against certain antagonistic and xenobiotic conditions encountered during pathogenic development.