Ribosomes account for a majority of the cell's RNA and much of its protein and represent a significant investment of cellular resources. The turnover and degradation of ribosomes has been proposed to play a role in homeostasis and during stress conditions. Mechanisms for the turnover of rRNA and ribosomal proteins have not been fully elucidated. We show here that the RNS2 ribonuclease and autophagy participate in RNA turnover in Arabidopsis thaliana under normal growth conditions. An increase in autophagosome formation was seen in an rns2-2 mutant, and this increase was dependent on the core autophagy genes ATG9 and ATG5. Autophagosomes and autophagic bodies in rns2-2 mutants contain RNA and ribosomes, suggesting that autophagy is activated as an attempt to compensate for loss of rRNA degradation. Total RNA accumulates in rns2-2, atg9-4, atg5-1, rns2-2 atg9-4, and rns2-2 atg5-1 mutants, suggesting a parallel role for autophagy and RNS2 in RNA turnover. rRNA accumulates in the vacuole in rns2-2 mutants. Vacuolar accumulation of rRNA was blocked by disrupting autophagy via an rns2-2 atg5-1 double mutant but not by an rns2-2 atg9-4 double mutant, indicating that ATG5 and ATG9 function differently in this process. Our results suggest that autophagy and RNS2 are both involved in homeostatic degradation of rRNA in the vacuole.

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.Keywords: Autophagy; degradation; stress; vacuole.

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation.

Localization of the RNase RNS2 to the vacuole via a C-terminal targeting signal is essential for its function in rRNA degradation and homeostasis. RNase T2 ribonucleases are highly conserved enzymes present in the genomes of nearly all eukaryotes and many microorganisms. Their constitutive expression in different tissues and cell types of many organisms suggests a housekeeping role in RNA homeostasis. The Arabidopsis thaliana class II RNase T2, RNS2, is encoded by a single gene and functions in rRNA degradation. Loss of RNS2 results in RNA accumulation and constitutive activation of autophagy, possibly as a compensatory mechanism. While the majority of RNase T2 enzymes is secreted, RNS2 is located within the vacuole and in the endoplasmic reticulum (ER), possibly within ER bodies. As RNS2 has a neutral pH optimum, and the endomembrane organelles are connected by vesicle transport, the site within the endomembrane system at which RNS2 functions is unclear. Here we demonstrate that localization to the vacuole is essential for the physiological function of RNS2. A mutant allele of RNS2, rns2-1, results in production of an active RNS2 RNase but with a mutation that removes a putative C-terminal vacuolar targeting signal. The mutant protein is, therefore, secreted from the cell. This results in a constitutive autophagy phenotype similar to that observed in rns2 null mutants. These findings illustrate that the intracellular retention of RNS2 and localization within the vacuole are critical for its cellular function.

Lack of rRNA recycling in rns2-2 cells triggers a change in carbon flux, which is redirected through the PPP to produce ribose-5-phosphate for de novo nucleoside synthesis. rRNA or ribosome turnover is thus essential for cellular homeostasis, probably through maintenance of nucleoside levels as part of the salvage pathway.

Ribonucleases belonging to the RNase T2 family are enzymes associated with the secretory pathway that are almost absolutely conserved in all eukaryotes. Studies in plants and vertebrates suggest they have an important housekeeping function in rRNA recycling. However, little is known about this family of enzymes in protostomes. We characterized RNase X25, the only RNase T2 enzyme in Drosophila melanogaster. We found that RNase X25 is the major contributor of ribonuclease activity in flies as detected by in gel assays, and has an acidic pH preference. Gene expression analyses showed that the RNase X25 transcript is present in all adult tissues and developmental stages. RNase X25 expression is elevated in response to nutritional stresses; consistent with the hypothesis that this enzyme has a housekeeping role in recycling RNA. A correlation between induction of RNase X25 expression and autophagy was observed. Moreover, induction of gene expression was triggered by oxidative stress suggesting that RNase X25 may have additional roles in stress responses. Phylogenetic analyses of this family in protostomes showed that RNase T2 genes have undergone duplication events followed by divergence in several phyla, including the loss of catalytic residues, and suggest that RNase T2 proteins have acquired novel functions. Among those, it is likely that a role in host immunosuppression evolved independently in several groups, including parasitic Platyhelminthes and parasitoid wasps. The presence of only one RNase T2 gene in the D. melanogaster genome, without any other evident secretory RNase activity detected, makes this organism an ideal system to study the cellular functions of RNase T2 proteins associated with RNA recycling and maintenance of cellular homeostasis. On the other hand, the discovery of gene duplications in several protostome genomes also presents interesting new avenues to study additional biological functions of this ancient family of proteins.

The soybean aphid (Aphis glycines) is one of the main insect pests of soybean (Glycine max) worldwide. Genomics approaches have provided important data on transcriptome changes, both in the insect and in the plant, in response to the plant-aphid interaction. However, the difficulties to transform soybean and to rear soybean aphid on artificial media have hindered our ability to systematically test the function of genes identified by those analyses as mediators of plant resistance to the insect. An efficient approach to produce transgenic soybean material is the production of transformed hairy roots using Agrobacterium rhizogenes; however, soybean aphids colonize leaves or stems and thus this approach has not been utilized. Here, we developed a hairy root system that allowed effective aphid feeding. We show that this system supports aphid performance similar to that observed in leaves. The use of hairy roots to study plant resistance is validated by experiments showing that roots generated from cotyledons of resistant lines carrying the Rag1 or Rag2 resistance genes are also resistant to aphid feeding, while related susceptible lines are not. Our results demonstrate that hairy roots are a good system to study soybean aphid-soybean interactions, providing a quick and effective method that could be used for functional analysis of the resistance response to this insect.

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