The light-harvesting chlorophyll a/b protein (LHCP) is nuclear-encoded and must traverse the chloroplast envelope before becoming integrally assembled into thylakoid membranes. Previous studies implicated a soluble stromal form of LHCP in the assembly pathway, but relied upon assays in which the thylakoid insertion step was intentionally impaired [Cline, K., Fulsom, D. R. and Viitanen, P. V. (1989) J. Biol. Chem. 264, 14225 -142321.Here we have developed a rapid-stopping procedure, based upon the use of HgC12, to analyze early events of the uninhibited assembly process. With this approach, we have found that proper assembly of LHCP into thylakoids lags considerably behind trans-envelope translocation. During the first few minutes of import, two distinct populations of mature-size LHCP accumulate within the chloroplast. One is the aforementioned soluble stromal intermediate, while the other is a partially (or improperly) assembled thylakoid species. Consistent with precursor/ product relationships, both species reach peak levels at a time when virtually none of the imported molecules are correctly assembled. These results confirm and extend our previous interpretation, that upon import, preLHCP is rapidly processed to its mature form, giving rise to a soluble stromal intermediate. They further suggest that the stromal intermediate initially inserts into the thylakoid bilayer in a partially assembled form, which eventually becomes properly assembled into the light-harvesting complex.In the last several years, targeting of nuclear-encoded chloroplast proteins has elicited widespread interest among molecular biologists and biochemists alike (see [l -31 for recent reviews). Similar to the situation for mitochondria, the information necessary for chloroplast recognition and translocation is largely contained in a cleavable N-terminal presequence [4-61, referred to as a transit peptide [7]. Generally speaking, chloroplast protein import can be divided into three steps: (a) binding of precursors to the outer chloroplast surface [8], presumably mediated by import receptors [9 -121, located at contact zones between the two envelope membranes [lo] ; (b) translocation across envelope membranes into the organelle; and finally, (c) intraorganellar sorting, which directs a given protein to its correct compartment. Furthermore, at some point during the overall process, the transit peptide is proteolytically removed, liberating a mature polypeptide. In contrast to mitochondria [13, 141, chloroplast protein import can be driven solely by ATP hydrolysis, without involvement of a protonmotive force [15-181. Recent evidence suggests that ATP is also required for the precursor-binding step [19].

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.

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