Gustavo C. MacIntosh

RNS2, a conserved member of the RNase T2 family, is necessary for ribosomal RNA decay in plants

RNase T2 enzymes are conserved in most eukaryotic genomes, and expression patterns and phylogenetic analyses suggest that they may carry out an important housekeeping role. However, the nature of this role has been elusive. Here we show that RNS2, an intracellular RNase T2 from Arabidopsis thaliana, is essential for normal ribosomal RNA recycling. This enzyme is the main endoribonuclease activity in plant cells and localizes to the endoplasmic reticulum (ER), ER-derived structures, and vacuoles. Mutants lacking RNS2 activity accumulate RNA intracellularly, and rRNA in these mutants has a longer half-life. Normal rRNA turnover seems essential to maintain cell homeostasis because rns2 mutants display constitutive autophagy. We propose that RNS2 is part of a process that degrades rRNA to recycle its components. This process appears to be conserved in all eukaryotes.

Constitutive and Induced Differential Accumulation of Amino Acid in Leaves of Susceptible and Resistant Soybean Plants in Response to the Soybean Aphid (Hemiptera: Aphididae)

ABSTRACT Although soybean aphid (Aphis glycines) resistance is commercially available in the form of the Rag1 gene, the mechanism of this resistance is not fully understood. Amino acids are a limiting factor for aphid growth, and there is evidence that plant amino acid composition is related to aphid resistance. Antibiotic resistance like that conferred by Rag1 could be associated in part with both protein and nonprotein free amino acids reducing survival, growth, and fecundity of the target pest. We posed two hypotheses: (1) A. glycines resistance is related to host quality in terms of free amino acids composition in the leaf, and (2) aphids may enhance host quality by inducing changes in the free amino acids composition. To test these hypotheses we conducted a field experiment using a split plot design, with soybean lines (a susceptible line and a related line carrying Rag1) as whole plots and aphid density as subplots (insecticide treated or left exposed to natural infestations). We analyzed free amino acids in leaves at three soybean developmental stages in all subplots. We observed significant whole and subplot effects on the concentration of a subset of amino acids tested. Susceptible and resistant plants had constitutive (whole-plot) differences in amino acids composition in all developmental stages analyzed. In addition, aphid-induced (subplot) responses of the plant to aphid infestation were found. We propose that the reduced nutritional quality of the resistant line and its reduced susceptibility to aphid-induced changes may contribute to aphid resistance conferred by Rag1.

Multiple Phytohormone Signals Control the Transcriptional Response to Soybean Aphid Infestation in Susceptible and Resistant Soybean Plants

The soybean aphid (Aphis glycines) is a major phloem-feeding pest of soybean (Glycine max). A. glycines feeding can cause the diversion of photosynthates and transmission of plant viruses, resulting in significant yield losses. In this study, we used oligonucleotide microarrays to characterize the long-term transcriptional response to soybean aphid colonization of two related soybean cultivars, one with the Rag1 aphid-resistance gene and one aphid-susceptible cultivar (without Rag1). Transcriptome profiles were determined after 1 and 7 days of aphid infestation. Our results revealed a susceptible response involving hundreds of transcripts, whereas only one transcript changed in the resistant response to aphids. This nonexistent resistance response might be explained by the fact that many defense-related transcripts are constitutively expressed in resistant plants, whereas these same genes are activated in susceptible plants only during aphid infestation. Analysis of phytohormone-related transcripts in the susceptible response showed different hormone profiles for the two time points, and suggest that aphids are able to suppress hormone signals in susceptible plants. A significant activation of abscissic acid, normally associated with abiotic stress responses, at day 7, might be a decoy strategy implemented by the aphid to suppress effective salicylic acid- and jasmonate-related defenses.

Performance and prospects of Rag genes for management of soybean aphid

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is an invasive insect pest of soybean [Glycine max (L.) Merr. (Fabaceae)] in North America, and it has led to extensive insecticide use in northern soybean‐growing regions there. Host plant resistance is one potential alternative strategy for managing soybean aphid. Several Rag genes that show antibiosis and antixenosis to soybean aphid have been recently identified in soybean, and field‐testing and commercial release of resistant soybean lines have followed. In this article, we review results of field tests with soybean lines containing Rag genes in North America, then present results from a coordinated regional test across several field sites in the north‐central USA, and finally discuss prospects for use of Rag genes to manage soybean aphids. Field tests conducted independently at multiple sites showed that soybean aphid populations peaked in late summer on lines with Rag1 or Rag2 and reached economically injurious levels on susceptible lines, whereas lines with a pyramid of Rag1 + Rag2 held soybean aphid populations below economic levels. In the regional test, aphid populations were generally suppressed by lines containing one of the Rag genes. Aphids reached putative economic levels on Rag1 lines for some site years, but yield loss was moderated, indicating that Rag1 may confer tolerance to soybean aphid in addition to antibiosis and antixenosis. Moreover, no yield penalty has been found for lines with Rag1, Rag2, or pyramids. Results suggest that use of aphid‐resistant soybean lines with Rag genes may be viable for managing soybean aphids. However, virulent biotypes of soybean aphid were identified before release of aphid‐resistant soybean, and thus a strategy for optimal deployment of aphid‐resistant soybean is needed to ensure sustainability of this technology.

What to Eat: Evidence for Selective Autophagy in Plants

Autophagy is a macromolecular degradation pathway by which cells recycle their contents as a developmental process, housekeeping mechanism, and response to environmental stress. In plants, autophagy involves the sequestration of cargo to be degraded, transport to the cell vacuole in a double-membrane bound autophagosome, and subsequent degradation by lytic enzymes. Autophagy has generally been considered to be a non-selective mechanism of degradation. However, studies in yeast and animals have found numerous examples of selective autophagy, with cargo including proteins, protein aggregates, and organelles. Recent work has also provided evidence for several types of selective autophagy in plants. The degradation of protein aggregates was the first selective autophagy described in plants, and, more recently, a hybrid protein of the mammalian selective autophagy adaptors p62 and NBR1, which interacts with the autophagy machinery and may function in autophagy of protein aggregates, was described in plants. Other intracellular components have been suggested to be selectively targeted by autophagy in plants, but the current evidence is limited. Here, we discuss recent findings regarding the selective targeting of cell components by autophagy in plants. [Formula: see text] [ Diane C. Bassham (Corresponding author)].

rnaset2 mutant zebrafish model familial cystic leukoencephalopathy and reveal a role for RNase T2 in degrading ribosomal RNA

T2-family acidic endoribonucleases are represented in all genomes. A physiological role for RNase T2 has yet to be defined for metazoa. RNASET2 mutation in humans is linked with a leukoencephalopathy that arises in infancy characterized by cortical cysts and multifocal white matter lesions. We now show localization of RNASET2 within lysosomes. Further, we demonstrate that loss of rnaset2 in mutant zebrafish results in accumulation of undigested rRNA within lysosomes within neurons of the brain. Further, by using high field intensity magnetic resonance microimaging, we reveal white matter lesions in these animals comparable to those observed in RNASET2-deficient infants. This correlates with accumulation of Amyloid precursor protein and astrocytes at sites of neurodegeneration. Thus we conclude that familial cystic leukoencephalopathy is a lysosomal storage disorder in which rRNA is the best candidate for the noxious storage material.

RNase T2 genes from rice and the evolution of secretory ribonucleases in plants

The plant RNase T2 family is divided into two different subfamilies. S-RNases are involved in rejection of self-pollen during the establishment of self-incompatibility in three plant families. S-like RNases, on the other hand, are not involved in self-incompatibility, and although gene expression studies point to a role in plant defense and phosphate recycling, their biological roles are less well understood. Although S-RNases have been subjects of many phylogenetic studies, few have included an extensive analysis of S-like RNases, and genome-wide analyses to determine the number of S-like RNases in fully sequenced plant genomes are missing. We characterized the eight RNase T2 genes present in the Oryza sativa genome; and we also identified the full complement of RNase T2 genes present in other fully sequenced plant genomes. Phylogenetics and gene expression analyses identified two classes among the S-like RNase subfamily. Class I genes show tissue specificity and stress regulation. Inactivation of RNase activity has occurred repeatedly throughout evolution. On the other hand, Class II seems to have conserved more ancestral characteristics; and, unlike other S-like RNases, genes in this class are conserved in all plant species analyzed and most are constitutively expressed. Our results suggest that gene duplication resulted in high diversification of Class I genes. Many of these genes are differentially expressed in response to stress, and we propose that protein characteristics, such as the increase in basic residues can have a defense role independent of RNase activity. On the other hand, constitutive expression and phylogenetic conservation suggest that Class II S-like RNases may have a housekeeping role.

Petunia nectar proteins have ribonuclease activity

Plants requiring an insect pollinator often produce nectar as a reward for the pollinators visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar.

The connection between ribophagy, autophagy and ribosomal RNA decay.

Ribosomes are essential components of all cells. A large body of knowledge has been accumulated regarding ribosome synthesis and assembly; however, the pathways of normal ribosome turnover, especially rRNA decay, are not known. Some information on ribosome recycling derives from studies on starved yeast cells that use a specialized type of autophagy, called ribophagy, to differentially target ribosomes for degradation. We found that Arabidopsis RNS2, a conserved ribonuclease of the RNase T2 family, is necessary for normal decay of rRNA. Mutants lacking RNS2 activity have longer-lived rRNA, accumulate RNA in the vacuole and show constitutive macroautophagy. Thus, it is clear that normal rRNA decay is necessary to maintain cellular homeostasis. These phenotypes and the subcellular localization of RNS2 in the endoplasmic reticulum and the vacuole suggest that RNS2 participates in a ribophagy-like mechanism that targets ribosomes for recycling under normal growth conditions.

Impact of transcriptional, ABA-dependent, and ABA-independent pathways on wounding regulation of RNS1 expression

Injured plants induce a wide range of genes whose products are thought to help to repair the plant or to defend against opportunistic pathogens that might infect the wounded plant. In Arabidopsis thaliana L., oligogalacturonides (OGAs) and jasmonic acid (JA) are the main regulators of the signaling pathways that control the local and systemic wound response, respectively. RNS1, a secreted ribonuclease, is induced by wounding in Arabidopsis independent of these two signals, thus indicating that another wound-response signal exists. Here we show that abscisic acid (ABA), which induces wound-responsive genes in other systems, also induces RNS1. In the absence of ABA signaling, wounding induces only approximately 45% of the endogenous levels of RNS1 mRNA. However, significant levels of RNS1 still accumulate in the absence of ABA signaling. Our results suggest that wound-responsive increases in ABA production may amplify induction of RNS1 by a novel ABA-independent pathway. To elucidate this novel pathway, we show here that the wound induction of RNS1 is due in part to transcriptional regulation by wounding and ABA. We also show evidence of post-transcriptional regulation which may contribute to the high levels of RNS1 transcript accumulation in response to wounding.