CsiLAC4 modulates boron flow in Arabidopsis and Citrus via high-boron-dependent lignification of cell walls.

The mechanisms underlying plant tolerance to boron (B) excess are far from fully understood. Here we characterized the role of the miR397-CsiLAC4/CsiLAC17 (from Citrus sinensis) module in regulation of B flow. Live-cell imaging techniques were used in localization studies. A tobacco transient expression system tested modulations of CsiLAC4 and CsiLAC17 by miR397. Transgenic Arabidopsis were generated to analyze the biological functions of CsiLAC4 and CsiLAC17. CsiLAC4's role in xylem lignification was determined by mRNA hybridization and cytochemistry. In situ B distribution was analyzed by laser ablation inductively coupled plasma mass spectrometry. CsiLAC4 and CsiLAC17 are predominantly localized in the apoplast of tobacco epidermal cells. Overexpression of CsiLAC4 in Arabidopsis improves the plants' tolerance to boric acid excess by triggering high-B-dependent lignification of the vascular system's cell wall and reducing free B content in roots and shoots. In Citrus, CsiLAC4 is expressed explicitly in the xylem parenchyma and is modulated by B-responsive miR397. Upregulation of CsiLAC4 in Citrus results in lignification of the xylem cell walls, restricting B flow from xylem vessels to the phloem. CsiLAC4 contributes to plant tolerance to boric acid excess via high-B-dependent lignification of cell walls, which set up a 'physical barrier' preventing B flow.

MicroRNA Sequencing Revealed Citrus Adaptation to Long-Term Boron Toxicity through Modulation of Root Development by miR319 and miR171.

Boron (B) toxicity in Citrus is a common physiological disorder leading to reductions in both productivity and quality. Studies on how Citrus roots evade B toxicity may provide new insight into plant tolerance to B toxicity. Here, using Illumina sequencing, differentially expressed microRNAs (miRNAs) were identified in B toxicity-treated Citrus sinensis (tolerant) and C. grandis (intolerant) roots. The results showed that 37 miRNAs in C. grandis and 11 miRNAs in C. sinensis were differentially expressed when exposed to B toxicity. Among them, miR319, miR171, and miR396g-5p were confirmed via 5'-RACE and qRT-PCR to target a myeloblastosis (MYB) transcription factor gene, a SCARECROW-like protein gene, and a cation transporting ATPase gene, respectively. Maintenance of SCARECROW expression in B treated Citrus roots might fulfill stem cell maintenance, quiescent center, and endodermis specification, thus allowing regular root elongation under B-toxic stress. Down-regulation of MYB due to up-regulation of miR319 in B toxicity-treated C. grandis roots might decrease the number of root tips, thereby dramatically changing root system architecture. Our findings suggested that miR319 and miR171 play a pivotal role in Citrus adaptation to long-term B toxicity by targeting MYB and SCARECROW, respectively, both of which are responsible for root growth and development.

Functional analysis of an alpha-1,2-mannosidase from Magnaporthe oryzae.

Identification of enzymes that are expressed during host colonization and characterization of their biochemical properties are prerequisite to understanding their role in the pathogen-host interaction. Nine alpha-1,2-mannosidase homologs were identified in the analysis of the Magnaporthe oryzae genome. Endoplasmic reticulum localized alpha-1,2-mannosidases play an important role in protein glycosylation. However, several members of the alpha-1,2-mannosidase gene family are predicted to be secreted. The biological role of such extracellular enzymes in host colonization has not been defined. Here, we characterized a secreted alpha-1,2-mannosidase of M. oryzae, MGG_00994.6, and found that the mature polypeptide is a glycoprotein capable of hydrolyzing alpha-1,2 linked mannobiose. The gene is expressed during growth in vitro and during colonization on rice plants, however, deletion of the gene did not affect pathogenicity. Five other members of the alpha-1,2-mannosidase of M. oryzae were expressed with a pattern similar to MGG_00994.6, suggesting the potential for functional redundancy. These results form the basis for additional studies on the role of this gene family in the rice blast fungus and its interaction with rice.

Biochemical and molecular characterization of a putative endoglucanase in Magnaporthe grisea.

Microbial pathogens secrete an array of cell wall-degrading enzymes to break down the structure of the host cell wall to facilitate colonization of the host tissue. To better understand their role in the pathogenesis, a putative endoglucanase from Magnaporthe grisea was characterized in this paper. SignalP-3.0 analysis indicates that the protein encoded by gene MGG_02532.5 in M. grisea (named MgEGL1 for M. grisea endoglucanase 1) contains a secretory signal peptide. Multiple alignment shows that MgEGL1 has high level of homology to endoglucanases (EC 3.1.1.4) from Aspergillus nidulans and Trichoderma reesei. The three proteins share a conserved catalytic domain, but only the one from T. reesei contains a cellulose binding module. MgEGL1 was constitutively expressed with the highest level in mycelia and the lowest in conidia. Interestingly, the MgEGL1 RNA could be alternatively processed when cultured in vitro and after infection of rice. Expression analysis confirmed that the MgEGL1 is a secreted protein. Its endoglucanase activity was assayed by Congo red plates, and further confirmed by the dinitrosalicylic acid method. The finding in this paper will provide the basis for further determination of the biochemical properties of the endoglucanase protein and its relevant function in fungal pathogenesis.