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Title:   Author  item
                                            Samac, Deborah

                                                         - *Debby*item
                                            Smigocki, Anna
  Submitted to: Phytopathology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 3/12/2003
                                    Publication  Date: 7/15/2003
                                    Citation: SAMAC, D.A., SMIGOCKI, A.C. EXPRESSION OF ORYZACYSTATIN I AND II IN ALFALFA INCREASES RESISTANCE TO THE ROOT-LESION NEMATODE. PHYTOPATHOLOGY. 2003. V. 93. P. 799-804.Interpretive Summary: Yields of alfalfa are significantly reduced due to feeding damage caused by insects and microscopic worms called nematodes. The guts of beetles and many nematodes contain digestive enzymes that break down proteins. These enzymes can be inactivated by specific proteins found in plants called cystatins. Because cystatins interact specifically with insect and nematode edigestive enzymes, they are attractive for controlling pests. The genes fo two cystatins from rice seeds and the gene for a marker enzyme were fused to a promoting DNA sequence so that the cystatins and the marker would be produced only in wounded cells, then the genes were introduced into alfalfa plants. Although the promoter is active only in wounded cells in potato and in rice, we found the marker enzyme present in unwounded alfalfa leaf and root cells. Wounding increased the amount of the marker enzyme in leaves about 2-fold over 24 hours. Feeding by nematodes increased the marker  enzyme at the feeding sites. Production of cystatins in roots reduced the populations of the root-lesion nematode 71-79% compared to plants without the cystatins. Some plants with cystatins had increased resistance to spring blackstem and leaf spot, a fungal disease of alfalfa. Genetic resistance to the root-lesion nematode and to spring black stem and leaf spot has been difficult to obtain. Both diseases reduce forage yields and decrease stand longevity because of damage to roots and crowns. These results show that cystatins have the potential to confer resistance to multiple diseases of alfalfa. Utilizing these genes in commercial alfalfa varieties would improve alfalfa yields and plant persistence and reduce the populations of root lesion nematodes that could infect subsequent crops.
                                Technical Abstract: Digestive cysteine proteinases have been detected in midguts of coleopteran and hemipteran insects and plant-parasitic nematodes. Phytocystatins, inhibitors of cysteine proteinases, are found in a number of plants where they may play a role in defense against pests and pathogens. The cDNAs of the phytocystatins from rice, oryzacystatin I (OC-I) and oryzacystatin II  (OC-II), were expressed in alfalfa (Medicago sativa) plants under the control of the potato protease inhibitor II (PinII) promoter and the plants evaluated for resistance to the root-lesion nematode (Pratylenchus penetrans) and to the fungal foliar pathogen, Phoma medicaginis. A PinII- beta-glucuronidase (GUS) gene was introduced into alfalfa to determine the pattern of gene expression from this promoter in alfalfa. Constitutive GUS expression was observed in leaf and root vascular tissue and in mesophyll of some plants. Mechanical wounding of leaves increased GUS expression  approximately 2-fold over 24 h. Inoculation with root-lesion or root-knot nematodes (Meloidogyne hapla) induced localized GUS expression near nematode feeding sites. Populations of root-lesion nematodes in alfalfa roots from 4 lines containing the OC-I transgene and 5 lines containing the OC-II transgene were reduced 71 to 79% compared to controls. One OC-II line consistently had significantly reduced leaf spot symptoms as compared to controls infected by P. medicaginis. These results indicate that oryzacystatins have the potential to confer multiple pest resistance in alfalfa.


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