Nicotiana benthamiana RanBP1-1 Is Involved in the Induction of Disease Resistance via Regulation of Nuclear-Cytoplasmic Transport of Small GTPase Ran.

Plant cells enhance the tolerances to abiotic and biotic stresses via recognition of the stress, activation and nuclear import of signaling factors, up-regulation of defense genes, nuclear export of mRNA and translation of defense proteins. Nuclear pore-mediated transports should play critical roles in these processes, however, the regulatory mechanisms of nuclear-cytoplasmic transport during stress responses are largely unknown. In this study, a regulator of nuclear export of RNA and proteins, NbRanBP1-1 (Ran-binding protein1-1), was identified as an essential gene for the resistance of Nicotiana benthamiana to potato blight pathogen Phytophthora infestans. NbRanBP1-1-silenced plants showed delayed accumulation of capsidiol, a sesquiterpenoid phytoalexin, in response to elicitor treatment, and reduced resistance to P. infestans. Abnormal accumulation of mRNA was observed in NbRanBP1-1-silenced plants, indicating that NbRanBP1-1 is involved in the nuclear export of mRNA. In NbRanBP1-1-silenced plants, elicitor-induced expression of defense genes, NbEAS and NbWIPK, was not affected in the early stage of defense induction, but the accumulation of NbWIPK protein was reduced. Nuclear export of the small G-protein NbRan1a was activated during the induction of plant defense, whereas this process was compromised in NbRanBP1-1-silenced plants. Silencing of genes encoding the nuclear pore proteins, Nup75 and Nup160, also caused abnormal nuclear accumulation of mRNA, defects in the nuclear export of NbRan1a, and reduced production of capsidiol, resulting in decreased resistance to P. infestans. These results suggest that nuclear export of NbRan is a key event for defense induction in N. benthamiana, and both RanBP1-1 and nucleoporins play important roles in the process.

Spatiotemporal deep imaging of syncytium induced by the soybean cyst nematode Heterodera glycines.

Parasite infections cause dramatic anatomical and ultrastructural changes in host plants. Cyst nematodes are parasites that invade host roots and induce a specific feeding structure called a syncytium. A syncytium is a large multinucleate cell formed by cell wall dissolution-mediated cell fusion. The soybean cyst nematode (SCN), Heterodera glycines, is a major soybean pathogen. To investigate SCN infection and the syncytium structure, we established an in planta deep imaging system using a clearing solution ClearSee and two-photon excitation microscopy (2PEM). Using this system, we found that several cells were incorporated into the syncytium; the nuclei increased in size and the cell wall openings began to be visible at 2 days after inoculation (DAI). Moreover, at 14 DAI, in the syncytium developed in the cortex, there were thickened concave cell wall pillars that resembled "Parthenon pillars." In contrast, there were many thick board-like cell walls and rarely Parthenon pillars in the syncytium developed in the stele. We revealed that the syncytia were classified into two types based on the pattern of the cell wall structures, which appeared to be determined by the position of the syncytium inside roots. Our results provide new insights into the developmental process of syncytium induced by cyst nematode and a better understanding of the three-dimensional structure of the syncytium in host roots.

Cell fusion and nuclear fusion in plants.

Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall.

Nucleoporin 75 is involved in the ethylene-mediated production of phytoalexin for the resistance of Nicotiana benthamiana to Phytophthora infestans.

Mature Nicotiana benthamiana shows stable resistance to the oomycete pathogen Phytophthora infestans. Induction of phytoalexin (capsidiol) production is essential for the resistance, which is upregulated via a mitogen-activated protein kinase (MAPK) cascade (NbMEK2-WIPK/SIPK) followed by ethylene signaling. In this study, NbNup75 (encodes a nuclear pore protein Nucleoporin75) was identified as an essential gene for resistance of N. benthamiana to P. infestans. In NbNup75-silenced plants, initial events of elicitor-induced responses such as phosphorylation of MAPK and expression of defense-related genes were not affected, whereas induction of later defense responses such as capsidiol production and cell death induction was suppressed or delayed. Ethylene production induced by either INF1 or NbMEK2 was reduced in NbNup75-silenced plants, whereas the expression of NbEAS (a gene for capsidiol biosynthesis) induced by ethylene was not affected, indicating that Nup75 is required for the induction of ethylene production but not for ethylene signaling. Given that nuclear accumulation of polyA RNA was increased in NbNup75-silenced plants, efficient export of mRNA from nuclei via nuclear pores would be important for the timely upregulation of defense responses. Collectively, Nup75 is involved in the induction of a later stage of defense responses, including the ethylene-mediated production of phytoalexin for the resistance of N. benthamiana to P. infestans.

Nicotiana benthamiana calreticulin 3a is required for the ethylene-mediated production of phytoalexins and disease resistance against oomycete pathogen Phytophthora infestans.

Mature Nicotiana benthamiana shows strong resistance to the potato late blight pathogen Phytophthora infestans. By screening using virus-induced random gene silencing, we isolated a gene for plant-specific calreticulin NbCRT3a as a required gene for resistance of N. benthamiana against P. infestans. NbCRT3a encodes an endoplasmic reticulum quality-control (ERQC) chaperone for the maturation of glycoproteins, including glycosylated cell-surface receptors. NbCRT3a-silenced plants showed no detectable growth defects but resistance to P. infestans was significantly compromised. Defense responses induced by the treatment with INF1 (a secretory protein of P. infestans), such as production of reactive oxygen species and accumulation of phytoalexins, were suppressed in NbCRT3a-silenced N. benthamiana. Expression of an ethylene-regulated gene for phytoalexin biosynthesis, NbEAS, was reduced in NbCRT3a-silenced plants, whereas the expression of salicylic acid-regulated NbPR-1a was not affected. Consistently, induction of ethylene production by INF1 was suppressed in NbCRT3a-silenced plants. Resistance reactions induced by a hyphal wall components elicitor prepared from P. infestans were also impaired in NbCRT3a-silenced plants. However, cell death induced by active mitogen-activated protein kinase kinase (NbMEK2(DD)) was not affected by the silencing of NbCRT3a. Thus, NbCRT3a is required for the initiation of resistance reactions of N. benthamiana in response to elicitor molecules derived from P. infestans.