Geminivirus Infection Up-Regulates the Expression of Two Arabidopsis Protein Kinases Related to Yeast SNF1- and Mammalian AMPK-Activating Kinases

Abstract

Geminivirus Rep-interacting kinase 1 (GRIK1) and GRIK2 constitute a small protein kinase family in Arabidopsis (Arabidopsis thaliana). An earlier study showed that a truncated version of GRIK1 binds to the geminivirus replication protein AL1. We show here both full-length GRIK1 and GRIK2 interact with AL1 in yeast two-hybrid studies. Using specific antibodies, we showed that both Arabidopsis kinases are elevated in infected leaves. Immunoblot analysis of healthy plants revealed that GRIK1 and GRIK2 are highest in young leaf and floral tissues and low or undetectable in mature tissues. Immunohistochemical staining showed that the kinases accumulate in the shoot apical meristem, leaf primordium, and emerging petiole. Unlike the protein patterns, GRIK1 and GRIK2 transcript levels only show a small increase during infection and do not change significantly during development. Treating healthy seedlings and infected leaves with the proteasome inhibitor MG132 resulted in higher GRIK1 and GRIK2 protein levels, whereas treatment with the translation inhibitor cycloheximide reduced both kinases, demonstrating that their accumulation is modulated by posttranscriptional processes. Phylogenetic comparisons indicated that GRIK1, GRIK2, and related kinases from Medicago truncatula and rice (Oryza sativa) are most similar to the yeast kinases PAK1, TOS3, and ELM1 and the mammalian kinase CaMKK, which activate the yeast kinase SNF1 and its mammalian homolog AMPK, respectively. Complementation studies using a PAK1/TOS3/ELM1 triple mutant showed that GRIK1 and GRIK2 can functionally replace the yeast kinases, suggesting that the Arabidopsis kinases mediate one or more processes during early plant development and geminivirus infection by activating SNF1-related kinases.

High-Frequency Reversion of Geminivirus Replication Protein Mutants during Infection

“…Nicotiana benthamiana plants were infected by bombardment or agroinoculation (16,42), while Arabidopsis thaliana Col-0 rosettes were infected by agroinoculation (66). For bombardment, wild-type or mutant replicon DNA (10 g) for either TGMV A or CaLCuV A was precipitated onto 1-mm gold microprojectiles in the presence of the corresponding wild-type B-replicon DNA.…”

Sugar Sensing and Signaling

“…These two protein kinases can complement a yeast mutant deficient in Snf1 upstream kinase activity (Hey et al, 2007). Interestingly, using a similar approach, the same upstream kinases, dubbed Geminivirus Rep-interacting kinase 1 and 2 (GRIK1/2), were isolated and shown to be induced in infected leaves, suggesting a role for SnRK1 in the biotic stress response (Shen and Hanley-Bowdoin, 2006). …”

“…Interestingly, a Snf1-like kinase gene is expressed asymmetrically in tomato apical meristems, with higher expression in the part destined to form leaf primordia, suggesting a role for SnRK1 in linking metabolism and energy supplies with meristematic activity (Pien et al, 2001). Future studies will likely reveal more roles of KIN10/11 in plant growth and development and in antiviral defense (Shen and Hanley-Bowdoin, 2006).…”

The SnRK1A Protein Kinase Plays a Key Role in Sugar Signaling during Germination and Seedling Growth of Rice

“…Recently, upstream kinases of SnRK1 were also identified in plants. Two Arabidopsis kinases, At SnAk1 and At SnAk2, were found to complement the yeast sak1 tos3 elm1 triple mutant and phosphorylate the Thr residue in the T-loop of SnRK1 (Shen and Hanley-Bowdoin, 2006;Hey et al, 2007). Plant SnRK1s, yeast SNF1, and mammalian AMPKs appear to be activated by different mechanisms: SnRK1s and SNF1 by sugars, and AMPKs by AMP .…”