To help learn how phytopathogens feed from their hosts, genes for nutrient transporters from the hemibiotrophic potato and tomato pest Phytophthora infestans were annotated. This identified 453 genes from 19 families. Comparisons with a necrotrophic oomycete, Pythium ultimum var. ultimum, and a hemibiotrophic fungus, Magnaporthe oryzae, revealed diversity in the size of some families although a similar fraction of genes encoded transporters. RNA-seq of infected potato tubers, tomato leaves, and several artificial media revealed that 56 and 207 transporters from P. infestans were significantly up- or down-regulated, respectively, during early infection timepoints of leaves or tubers versus media. About 17 were up-regulated >4-fold in both leaves and tubers compared to media and expressed primarily in the biotrophic stage. The transcription pattern of many genes was host-organ specific. For example, the mRNA level of a nitrate transporter (NRT) was about 100-fold higher during mid-infection in leaves, which are nitrate-rich, than in tubers and three types of artificial media, which are nitrate-poor. The NRT gene is physically linked with genes encoding nitrate reductase (NR) and nitrite reductase (NiR), which mobilize nitrate into ammonium and amino acids. All three genes were coregulated. For example, the three genes were expressed primarily at mid-stage infection timepoints in both potato and tomato leaves, but showed little expression in potato tubers. Transformants down-regulated for all three genes were generated by DNA-directed RNAi, with silencing spreading from the NR target to the flanking NRT and NiR genes. The silenced strains were nonpathogenic on leaves but colonized tubers. We propose that the nitrate assimilation genes play roles both in obtaining nitrogen for amino acid biosynthesis and protecting P. infestans from natural or fertilization-induced nitrate and nitrite toxicity.

With the rapid development of micro total analysis systems and sensitive biosensing technologies, it is often desirable to immobilize biomolecules to small areas of surfaces other than silicon. To this end, photolithographic techniques were used to derivatize micrometer-sized, spatially segregated biosensing elements on several different substrate surfaces. Both an interference pattern and a dynamic confocal patterning apparatus were used to control the dimensions and positions of immobilized regions. In both of these methods, a UV laser was used to initiate attachment of a photoactive biotin molecule to the substrate surfaces. Once biotin was attached to a substrate, biotin/avidin/biotin chemistry was used to attach fluorescently labeled or nonlabeled avidin and biotinylated sensing elements such as biotinylated antibodies. Dimensions of 2-10 microm were achievable with these methods. A wide variety of materials, including glassy carbon, quartz, acrylic, polystyrene, acetonitrile-butadiene-styrene, polycarbonate, and poly(dimethylsiloxane), were used as substrates. Nitrene- and carbene-generating photolinkers were investigated to achieve the most homogeneous films. These techniques were applied to create a prototype microfluidic sensor device that was used to separate fluorescently labeled secondary antibodies.

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A m i c r o f l u i d i c c h i p has been developed which allows for t h e d i r e c t d e t e c t i o n of several d i s t i n c t DNA t a r g e t s present i n a sample (Figure 1). The d i r e c t detection o f underivatized DNA i s advantageous f o r t h e analysis o f very small amounts o f sample, where contamination i s a major concern. DNA probes can be attached i n microchannels present on t h e c h i p v i a a b i o t i d a v i d i n linkage. .The sample i s then introduced i n t o t h e channel where an appropriate DNA t a r g e t hybridizes w i t h i t s complementary probe. Following h y b r i d i z a t i o n o f t h e t a r g e t , an a l k a l i n e b u f f e r i s introduced t o t h e channel t o dehybridize t h e double-stranded DNA and f l u s h t h e t a r g e t downstream t o a copper electrode. The dehybridized DNA i s then detected electrochemically. The e l u t i o n time can be used t o i d e n t i f y t h e p a r t i c u l a r DNA t a r g e t since t h e DNA probes are s p a t i a l l y segregated i n t h e channel (Figure 2 ) . I n t e g r a t i n g t h e detector and t h e sensing probes on t h e m i c r o f l u i d i c c h i p allows f o r an inexpensive and e a s i l y f a b r i c a t e d biosensor device f o r t h e precise r e c o g n i t i o n and subsequent d e t e c t i o n o f a s p e c i f i c complimentary DNA t a r g e t f o r diagnosis and genetic screeni ng . Electrochemical d e t e c t i o n i s capable o f working w i t h small samples making i t a f i t t i n g technique f o r DNA analysis.' Copper surfaces have been found t o c a t a l y z e t h e o x i d a t i o n o f t h e sugar backbone o f DNA w i t h o u t f o u l i n g . Therefore, sinusoidal voltammetry was used t o detect t h e separated DNA fragments as they e l u t e d from t h e channel. A l a r g e amplitude s i n e wave ( > 50mV) was used as t h e e x c i t a t i o n waveform, such t h a t t h e f a r a d a i c signal i s pushed out t o t h e higher harmonics w h i l e t h e background c u r r e n t remains a t t h e fundamental frequency.'. U t i l i z a t i o n of t h e frequency domain i n t h i s manner increases t h e s i g n a l t o noise of t h e measurement a t t h e higher harmonics, a1 lowing t h e d e t e c t i o n of picomolar concentrations o f DNA.